Immunoconjugates targeting cd138 and uses thereof

ABSTRACT

Disclosed are immunoconjugates having in particular specificity for CD138 expressed on target cells and which display homogenous targeting. The immunoconjugates may be sterially hindered and/or contain a cleavable linker.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 12/342,407, filed Dec. 23, 2008, which claims the benefit of U.S.provisional application 61/016,620, filed Dec. 26, 2007, provisionalapplication 61/087,466, file Aug. 8, 2008 and provisional application61/087,590, also filed on Aug. 8, 2008, all of which are incorporatedherein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to improved targeting agents for theantigen CD138, immunoconjugates comprising such targeting agents,compositions comprising the immunoconjugates and methods employing them.

BACKGROUND

CD138, which acts as a receptor for the extracellular matrix, isoverexpressed on multiple myeloma (MM) cells and has been shown toinfluence MM cell development and/or proliferation. CD138 is alsoexpressed on cells of ovarian carcinoma, kidney carcinoma, gall bladdercarcinoma, breast carcinoma, prostate cancer, lung cancer, coloncarcinoma cells and cells of Hodgkin's and non-Hodgkin's lymphomas,chronic lymphocytic leukemia (CLL) to name just a few.

The publications and other materials, including patents, used herein toillustrate the invention and, in particular, to provide additionaldetails respecting the practice are incorporated by reference. Forconvenience, the publications are referenced in the following text byauthor and date and/or are listed alphabetically by author in theappended bibliography.

Tassone et al. (2004) have reported excellent binding of the murine IgG1antibody B-B4 to the CD138 antigen expressed on the surface of MM cells.Tassone also reported high cytotoxic activity of the immunoconjugateB-B4-DM1, which comprises the mytansinoid DM1 as an effector molecule,against multiple myeloma cells (see also US Patent Publ. 20070183971).

While Tassone et al. have contributed to providing an effectivetreatment of MM and a composition of matter that may be employed in sucha treatment, there remain a number of needs in the art.

There remains a need for immunoconjugates based on B-B4 that are devoidof certain properties and/or functions associated with B-B4. There is,in particular a need for a chimerized antibody based on B-B4 that bindsthe CD138 as effectively as B-B4 but can be administered to humanswithout significant side effects. There is also a need for such a B-B4based immunoconjugate that shows one or more advantageous propertiesrelative to its murine counterpart. Those properties include improvedantigen binding, improved killing of tumor cells comprising, inparticular of CD138 expressing tumor cells, and cells accessory theretoor more homogenous binding of the target.

SUMMARY OF THE INVENTION

The present invention is directed at an immunoconjugate comprising:

(a) an engineered targeting antibody, and(b) an effector molecule,wherein said immunoconjugate homogenously targets CD138 expressingtarget cells.

The engineered targeting antibody of the present invention may

(i) consist essentially of antigen binding region (ABR) against CD138 ofa non-human antibody, or

(ii) comprise an antigen binding region (ABR) against CD138, whereinsaid antigen binding region is of a non-human antibody, and

a further antibody region, wherein at least part of said furtherantibody region is of a human antibody.The ABR may comprise:(a) heavy chain variable region CDR3 comprising amino acid residues 99to 111 of SEQ ID NO: 1, and(b) light chain variable region CDR3 comprising amino acid residues 89to 97 of SEQ ID NO: 2, respectively.The ABR may further comprise:(a) heavy chain variable region CDR1 and CDR2 comprising amino acidresidues 31 to 35 and 51 to 68 of SEQ ID NO: 1, and/or(b) light chain variable region CDR1 and CDR 2 comprising amino acidresidues 24 to 34 and 50 to 56 of SEQ ID NO: 2, respectively.The further antibody region may comprise:(a) amino acid residues 123 to 448 of SEQ ID NO: 1, and/or(b) amino acid residues 108 to 214 of SEQ ID NO: 2, respectivelyand mutations thereof that

(i) maintain or lower the antibody-dependent cytotoxicity and/orcomplement-dependent cytotoxicity of the engineered targeting antibodyand/or

(ii) stabilize the engineered targeting antibody.

The effector molecule may be attached to said engineered targetingantibody via a linker. The linker may comprise a disulfide bond. Theeffector molecule (e.g., DM4) may provide sterical hindrance between thetargeting antibody and the effector molecule. The effector molecule maybe at least one maytansinoid (e.g., DM1, DM3, or DM4) taxane or aCC1065, or an analog thereof.

The immunoconjugate may bind CD138 with a targeting variation of lessthan 150%, 140%, 130%, 120%, 110%, 100%, 90%, 80%, 70%, 60% or 50%.

The present invention is also directed at an immunoconjugate comprising:

a targeting agent targeting CD138 comprising

an isolated polypeptide comprising an amino acid sequence of animmunoglobulin heavy chain or part thereof, wherein said immunoglobulinheavy chain or part thereof has at least 70% sequence identity with SEQID NO:1. A constant region of said immunoglobulin heavy chain or saidpart thereof may be an IgG4 isotype constant region.

The present invention is also directed at a method of treating MM in asubject, comprising:

providing one of more of the immunoconjugates specified herein, andadministering to said subject said immunoconjugate in an amounteffective to treat multiple myeloma.

The targeting agent of the immunoconjugate may comprise a light chainsequence having at least about 70% sequence identity with SEQ ID NO:2.The targeting agent of the immunoconjugate may also comprise a heavychain sequence having at least about 70% sequence identity with SEQ IDNO:1.

The present invention is also directed at a method for immunoconjugatemediated drug delivery comprising:

providing one or more of the immunoconjugates specified herein, andadministering said immunoconjugate in a therapeutically effectiveamount, wherein said IgG4 isotype alleviates ADCC, complement dependentcytotoxicity and/or Fc-mediated targeting of hepatic FcR.

The present invention is also directed at a method for inhibiting,delaying and/or preventing the growth of tumor cells in a cell culturecomprising

administering to said cell culture a growth of tumor cells inhibiting,delaying and/or preventing effective amount of one or more of theimmunoconjugates specified herein. The effective amount may induce celldeath or continuous cell cycle arrest in CD138 expressing tumor cellsand, optionally, auxiliary cells that do not express CD138, inparticular tumor stroma cells. The cells in said cell culture may beobtained from a cancer patient and, after administration of saideffective amount of said immunoconjugate, the cells of said cell culturemay be reimplanted into said cancer patient.

The present invention is also directed at a method for inhibiting,delaying and/or preventing the growth of a tumor comprising CD138 tumorcells and/or spread of tumor cells of such a tumor in a patient in needthereof, comprising

administering to said patient at least one or more of theimmunoconjugates specified above in a growth of said tumor and/orspreading of said tumor cells inhibiting or reducing amount,

-   -   wherein said immunoconjugate inhibits, delays or prevents the        growth and/or spread of said tumor cells.

The effector molecule of said immunoconjugate(s) may be a toxin,cytotoxic enzyme, low molecular weight cytotoxic drug, a pore-formingagent, biological response modifier, prodrug activating enzyme, anantibody, cytokine or a radionuclide.

Immunoconjugates of the present invention may be administered in asingle dose of 5 mg/m² to about 300 mg/m², optionally at hourly, daily,weekly intervals or combinations thereof.

Multiple dose regimes include, hourly, daily and weekly regimes are partof the present invention and include in particular administration atintervals of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23 hours, 1, 2, 3, 4, 5, 6, 7 days, 1, 2, 3, 4, 5, 6, 7 or 8weeks.

The present invention is also directed at a method for inhibiting,delaying and/or preventing the growth of a tumor and/or spread ofmalignant tumor cells in a patient in need thereof, comprising

(a) administering to said patient one or more cytotoxic agents and/orradiation in an amount effective to reduce tumor load; and

(b) administering to said patient at least one of the immunconjugatesspecified herein in a growth of a tumor and/or spreading of tumor cellsinhibiting, delaying or preventing amount,

-   -   wherein said immunoconjugate inhibits, delays or prevents the        growth and/or spread of tumor cells comprising CD138 expressing        cells.

The cytotoxic agent may, in particular, be mephalan, vincristine,doxorubicin, dexamethasone, cyclophosphamide, etoposide, cytarabine,cisplatin, thalidomide, prednisone, thalidomide, bortezomib,lenalidomide, sorafenib, romidepsin or combinations thereof or may beantibody based.

The present invention is also directed at a method for treating asubject having a condition that would benefit from the suppression ofmyeloma cell survival, the method comprising:

(a) providing at least one of any of the immunoconjugates specifiedherein, and(b) administering the immunoconjugate to the subject to selectivelydecrease survival or growth of said myeloma cells of said subject.

The present invention is also directed at a pharmaceutical compositioncomprising any of the immunoconjugates specified herein for theinhibition, delay and/or prevention of the growth of tumors and/orspread of tumor cells, and one or more pharmaceutically acceptableexcipients.

The pharmaceutical composition may include cytotoxic agents as specifiedherein.

The present invention is also directed at a kit comprising, in separatecontainers, pharmaceutical compositions for use in combination toinhibit, delay and/or prevent the growth of tumors and/or spread oftumor cells, wherein one container comprises an effective amount of theabove pharmaceutical composition, and wherein, a separate containercomprises a second pharmaceutical composition comprising an effectiveamount of an agent, preferably a cytotoxic agent, for the inhibition,delay and/or prevention of the growth of tumors and/or spread of tumorcells, and one or more pharmaceutically acceptable excipients.

The present invention is also directed at a method for inhibiting,delaying and/or preventing growth of a tumor comprising CD138 tumorcells and/or spread of tumor cells of such a tumor in a subject in needthereof, comprising

(a) providing an immunoconjugate comprising:an engineered targeting antibody against CD138 attached to an effectormolecule via a cleavable linker, wherein said effector molecule issterically hindered, and(b) administering to said subject the immunoconjugate of (a) in a growthof said tumor and/or spreading of said tumor cells inhibiting, delayingand/or preventing amount, wherein said immunoconjugate of (a) provides agrowth of a tumor inhibiting activity that exceeds that of itsunhindered counterpart by about 10%, about 20%, about 30%, about 40% ormore.

A growth of a tumor inhibiting activity of an unhindered counterpartcomprising a non-cleavable linker may exceed that of the growth of atumor inhibiting activity of its unhindered counterpart comprising acleavable linker, such as by at least about 5%, at least about 10%, upto about 15%.

Said engineered targeting antibody against CD138 may consist essentiallyof antigen binding region against CD138 of a non-human antibody or maycomprise an antigen binding region against CD138 of a non-human antibodyand a further antibody region, wherein at least part of said furtherantibody region is of a human antibody.

Said cleavable linker may comprise a disulfide bond. The effectormolecule may be DM4. The immunoconjugate may be part of a pharmaceuticalcomposition and may be administered to the subject in at least one dosein an amount from about 5 mg/m² to about 300 mg/m².

The present invention provides an immunoconjugate for use as amedicament wherein the immunoconjugate comprises:(a) an engineered targeting antibody

(i) consisting essentially of antigen binding region against CD138 of anon-human antibody, or

(ii) comprising an antigen binding region against CD138, wherein saidantigen binding region is of a non-human antibody,

a further antibody region, wherein at least part of said furtherantibody region is of a human antibody, and(b) an effector molecule,wherein said immunoconjugate homogenously binds to CD138.The present invention provides a further immunoconjugate for use as amedicament comprising:

a targeting agent targeting CD138 comprising

an isolated polypeptide comprising an amino acid sequence of animmunoglobulin heavy chain or part thereof, wherein said immunoglobulinheavy chain or part thereof has at least 70% sequence identity with SEQID NO:1.

In particular, in one aspect of the invention the immunoconjugate of theabove paragraph is for use in the treatment of multiple myeloma. Inparticular, the immunoconjugate can be used for the manufacture of amedicament for the treatment of multiple myeloma.

The present invention further provides an immunoconjugate for use inimmunoconjugate mediated drug delivery to a patient, in particular foralleviation of ADCC, complement dependent cytotoxicity and/orFc-mediated targeting of hepatic FcR, wherein the immunoconjugatecomprises a targeting agent targeting CD138 comprising an isolatedpolypeptide comprising an amino acid sequence of an immunoglobulin heavychain or part thereof, wherein said immunoglobulin heavy chain or partthereof has at least 70% sequence identity with SEQ ID NO:1, and whereina constant region of said immunoglobulin heavy chain or part thereof isan IgG4 isotype constant region.

The present invention also provides tumor cells for use in the treatmentof cancer in a patient wherein the tumor cells have been treated in cellculture with an immunoconjugate comprising:(a) an engineered targeting antibody

(i) consisting essentially of antigen binding region against CD138 of anon-human antibody, or

(ii) comprising an antigen binding region against CD138, wherein saidantigen binding region is of a non-human antibody,

a further antibody region, wherein at least part of said furtherantibody region is of a human antibody, and(b) an effector molecule,wherein said immunoconjugate homogenously binds to CD138.The present invention also provides tumor cells for use in the treatmentof cancer in a patient wherein the tumor cells have been treated in cellculture with an immunoconjugate comprising:

a targeting agent targeting CD138 comprising

an isolated polypeptide comprising an amino acid sequence of animmunoglobulin heavy chain or part thereof, wherein said immunoglobulinheavy chain or part thereof has at least 70% sequence identity with SEQID NO:1.

The present invention provides an immunoconjugate for use in inhibiting,delaying and/or preventing the growth of a tumor comprising CD138 tumorcells and/or spread of tumor cells of such a tumor in a patient, whereinthe immunoconjugate comprises:(a) an engineered targeting antibody

(i) consisting essentially of antigen binding region against CD138 of anon-human antibody, or

(ii) comprising an antigen binding region against CD138, wherein saidantigen binding region is of a non-human antibody,

a further antibody region, wherein at least part of said furtherantibody region is of a human antibody, and(b) an effector molecule,wherein said immunoconjugate homogenously binds to CD138.Alternatively, the present invention provides an immunoconjugate for usein inhibiting, delaying and/or preventing the growth of a tumorcomprising CD138 tumor cells and/or spread of tumor cells of such atumor in a patient, wherein the immunoconjugate comprises:

a targeting agent targeting CD138 comprising

an isolated polypeptide comprising an amino acid sequence of animmunoglobulin heavy chain or part thereof, wherein said immunoglobulinheavy chain or part thereof has at least 70% sequence identity with SEQID NO:1.

Still further, the present invention provides a medicament comprising animmunoconjugate and one or more cancer drugs as a combined preparationfor simultaneous, separate or sequential use in the treatment of tumorcells comprising CD138 expressing cells, wherein the immunoconjugatecomprises:(a) an engineered targeting antibody

(i) consisting essentially of antigen binding region against CD138 of anon-human antibody, or

(ii) comprising an antigen binding region against CD138, wherein saidantigen binding region is of a non-human antibody,

a further antibody region, wherein at least part of said furtherantibody region is of a human antibody, and(b) an effector molecule,wherein said immunoconjugate homogenously binds to CD138,and wherein the one or more cancer drugs are capable of reducing thetumor load.Alternatively, the present invention provides a medicament comprising animmunoconjugate and one or more cancer drugs as a combined preparationfor simultaneous, separate or sequential use in the treatment of tumorcells comprising CD138 expressing cells, wherein the immunoconjugatecomprises:

a targeting agent targeting CD138 comprising

an isolated polypeptide comprising an amino acid sequence of animmunoglobulin heavy chain or part thereof, wherein said immunoglobulinheavy chain or part thereof has at least 70% sequence identity with SEQID NO:1,

and wherein the one or more cancer drugs are capable of reducing thetumor load.In a further aspect of the use of the above two paragraphs the combinedpreparation is to be administered to a patient who has been treated withradiation.In an alternative aspect the present invention provides the use of animmunoconjugate for the manufacture of a medicament for treating tumorcells in a patient comprising CD138 expressing cells, wherein theimmunoconjugate comprises:(a) an engineered targeting antibody

(i) consisting essentially of antigen binding region against CD138 of anon-human antibody, or

(ii) comprising an antigen binding region against CD138, wherein saidantigen binding region is of a non-human antibody,

a further antibody region, wherein at least part of said furtherantibody region is of a human antibody, and(b) an effector molecule,wherein said immunoconjugate homogenously binds to CD138,and wherein the medicament is to be administered to a patient treatedwith radiation to reduce the tumor load.Still further the present invention provides the use of animmunoconjugate for the manufacture of a medicament for treating tumorcells in a patient comprising CD138 expressing cells, wherein theimmunoconjugate comprises:

a targeting agent targeting CD138 comprising

an isolated polypeptide comprising an amino acid sequence of animmunoglobulin heavy chain or part thereof, wherein said immunoglobulinheavy chain or part thereof has at least 70% sequence identity with SEQID NO:1,

and wherein the medicament is to be administered to a patient treatedwith radiation to reduce the tumor load.In the above paragraphs, the medicament is capable of inhibiting,delaying and/or preventing the growth of a tumor and/or spread ofmalignant tumor cells in a patient.Further the present invention provides an immunoconjugate forsuppression of myeloma cell survival in an individual wherein theimmunoconjugate comprises:(a) an engineered targeting antibody

(i) consisting essentially of antigen binding region against CD138 of anon-human antibody, or

(ii) comprising an antigen binding region against CD138, wherein saidantigen binding region is of a non-human antibody,

a further antibody region, wherein at least part of said furtherantibody region is of a human antibody, and(b) an effector molecule,wherein said immunoconjugate homogenously binds to CD138.Still further the present invention provides an immunoconjugate forsuppression of myeloma cell survival in an individual wherein theimmunoconjugate comprises:

a targeting agent targeting CD138 comprising

an isolated polypeptide comprising an amino acid sequence of animmunoglobulin heavy chain or part thereof, wherein said immunoglobulinheavy chain or part thereof has at least 70% sequence identity with SEQID NO:1.

In the above two paragraphs the immunoconjugate is, in particular,capable of selectively decreasing the survival or growth of said myelomacells in the individual.Further, the present invention provides an immunoconjugate for use ininhibiting, delaying and/or preventing growth of a tumor comprisingCD138 tumor cells and/or spread of tumor cells of such a tumor in asubject wherein the immunoconjugate comprises an engineered targetingantibody against CD138 attached to an effector molecule via a cleavablelinker, wherein said effector molecule is sterically hindered.In the above paragraph, the immunoconjugate is, in particular, capableof providing a tumor growth inhibiting activity that exceeds that of itsunhindered counterpart by about 10%, about 20%, about 30%, about 40% ormore.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a schematic representation of nBT062 having effectormolecules attached.

FIG. 2 is a chemical representation of BT062.

FIG. 3 shows the conversion of ansamitocin P-3 to maytansinol(stereochemistry is omitted for simplicity).

FIG. 4 shows a representative synthesis scheme of DM4.

FIG. 5 is a schematic representation of an antibody conjugation (nBT062to DM4).

FIG. 6 shows an analysis of the binding of nBT062-SPDB-DM4,nBT062-SPP-DM1, nBT062-SMCC-DM1 and nBT062 antibody to OPM-2 cells.Different concentrations of nBT062 and conjugates were given to thecells and mean fluorescence was measured by FACS analysis.

FIG. 7(A)-(D) depict in vitro cytotoxicity of nBT062-DMx conjugatestowards MOLP-8 (CD138⁺) and BJAB (CD138⁻) cells. The cells were culturedin flat bottom plates and incubated with the indicated concentrations ofimmunoconjugates for 5 days. In FIG. 7(A), nBT062-DMx isnBT062-SPDB-DM4, in FIG. 7(B), nBT062-DMx is nBT062-SPP-DM1, in FIG.7(C), nBT062-DMx is nBT062-SMCC-DM1 and in in FIG. 7(D), nBT062-DMx isnBT062-SPDB-DM4. WST reagent was added for further 3 hours to assesscell viability. In FIG. 7(D) cytotoxic activity of nBT062-SPDB-DM4 wasanalyzed in the presence or absence of blocking antibody (1 μM nBT062).

FIGS. 8 (A)-(D) show tumor volumes for individual mice treated with (A)PBS (FIG. 8(A)), (B) nBT062 antibody (FIG. 8(B)), (C) free DM4 (FIG.8(C)) or (D) non-targeting conjugate huC242-DM4 (FIG. 8(D)) over time(days) post-inoculation with MOLP-8 tumor cells.

FIG. 9 (A)-(D) show tumor volumes for individual mice treated with (A)PBS (FIG. 9(A)), (B) nBT062-SPDB-DM4 (FIG. 9(B)), (C) B-B4-SPP-DM1 (FIG.9(C)) or (D) nBT062-SPP-DM1 (FIG. 9(D)) over time (days)post-inoculation with MOLP-8 tumor cells.

FIG. 10 depicts mean tumor volume (+/−SD) of MOLP-8 human multiplemyeloma xenografts in CB.17 SCID mice over time (days) post-inoculation.

FIG. 11(A) and FIG. 11(B) show the anti-tumor activity of nBT062-DMxagainst CD138⁺ MOLP-8 tumor cells in a bulky MOLP-8 tumor model in SCIDmice. Tumor volume is given as mean (+/−SD) for each group. The group inFIG. 11(A) comprises 8 mice, the group in FIG. 11(B) comprises 4 mice.

FIG. 12 is a graph reflecting the anti-tumour efficacy of nBT062containing DMx conjugates in the SCIDhu/INA-6 model towards multiplemyeloma cells in the environment of human bone marrow. Soluble humanIL-6 Receptor produced by multiple myeloma cells (shuIL-6R) was used asan indicator for tumor burden. Triangle: nBT062-SPP-DM1, Square:nBT062-SPDB-DM4; Diamond: vehicle control.

FIG. 13 shows nBT062-SPDB-DM4 mediated bystander killing in vitro. CD138positive OPM2 cells and CD138 negative Namawla cells were cultured withnBT062-SPDB-DM4 at different concentrations and cell viablility wasmeasured. OD₄₅₀ values represent a measure for cell viability.

DETAILED DESCRIPTION OF VARIOUS AND PREFERRED EMBODIMENTS OF THEINVENTION

The present invention relates to immunoconjugates comprising CD138targeting agents and the delivery of the effector molecule(s) of theimmunoconjugates to target sites and the site specific release ofeffector(s) molecule in, at or near target cells, tissues and organs.More particularly, the present invention relates to immunoconjugatescomprising CD138 targeting agents and potent effector molecules that areattached to the targeting agent. The effector molecules may be activatedby cleavage/dissociation from the targeting agent portion of theimmunoconjugate at the target site.

The immunoconjugates according to the present invention may beadministered to a subject in need of therapeutic treatment or to cellsisolated from such a subject in need of therapeutic treatment. Theeffector molecule or molecules may be released from the immunoconjugateby cleavage/dissociation in, at or close to the target cell, tissue ororgan.

In one example, the immunoconjugate comprises the antibody nBT062, whichtargets CD138 expressing cells, and at least one highly cytotoxic drugor toxin as an effector molecule, is administered to a patient withcancer. In this example, a therapeutically effective amount of theimmunoconjugate is administered intravenously to a patient so that itconcentrates in the cancer cells. The effector molecule or molecules arethen released from the antibody by natural means. After or duringcleavage the effector molecule may be stabilized by alkylation and maydiffuse to surrounding auxiliary cells such as stroma cells that do notexpress CD138.

In a second example, the immunoconjugate comprises the antibody nBT062,which targets CD138 expressing cells, and at least one highly cytotoxicdrug or toxin as an effector molecule, and an additional cytotoxic agentis administered to a patient with cancer. In this example, atherapeutically effective amount of the immunoconjugate and thecytotoxic agent are co-administered intravenously to a patient so thatit concentrates in the cancer cells. The cytotoxic agent destroys morethan 50% of the CD138 expressing cancer cells, but the immunconjugateattaches efficiently to further CD138 expressing cancer cells. Theeffector molecule or molecules are released from the antibody by naturalmeans. After or during cleavage, the effector molecule may be stabilizedby alkylation and may diffuse to surrounding auxiliary cells such asstroma cells that do not express CD138.

In a third example, the immunoconjugate comprises the antibody nBT062and at least one highly cytotoxic drug or toxin and is administered to acell population isolated from a patient with cancer. In this example, acell death or continuous cell cycle arrest inducing amount of theimmunoconjugate is administered to the cell population so that itconcentrates in the cancerous cells. The effector molecule or moleculesare released from the targeting antibody by natural means or externalmeans to induce cell death or continuous cell cycle arrest in the cancercells.

In a fourth example, the immunoconjugate comprises the antibody nBT062and at least one highly cytotoxic drug or toxin as an effector moleculeand is administered to a patient with cancer. In this example, atherapeutically effective amount of the immunoconjugate is administeredintravenously to a patient so that it concentrates in the cancerouscells. The effector molecule or molecules are released from the antibodytarget by an external means to induce cell death or continuous cellcycle arrest in the cancer cells.

CD138 or syndecan-1 (also described as SYND1; SYNDECAN; SDC; SCD1; CD138ANTIGEN, SwissProt accession number: P18827 human) is a membraneglycoprotein that was originally described to be present on cells ofepithelial origin, and subsequently found on hematopoietic cells(Sanderson, 1989). CD138 has a long extracellular domain that binds tosoluble molecules (e.g., the growth factors EGF, FGF, HGF) and toinsoluble molecules (e.g., to the extracellular matrix componentscollagen and fibronectin) through heparan sulfate chains (Langford,1998; Yang, 2007) and acts as a receptor for the extracellular matrix.CD138 also mediates cell to cell adhesion through heparin-bindingmolecules expressed by adherent cells. It has been shown that CD138 hasa role as a co-receptor for growth factors of myeloma cells (Bisping,2006). Studies of plasma cell differentiation showed that CD138 mustalso be considered as a differentiation antigen (Bataille, 2006).

In malignant hematopoiesis, CD138 is highly expressed on the majority ofMM cells, ovarian carcinoma, kidney carcinoma, gall bladder carcinoma,breast carcinoma, prostate cancer, lung cancer, colon carcinoma cellsand cells of Hodgkin's and non-Hodgkin's lymphomas, chronic lymphocyticleukemia (CLL) (Horvathova, 1995), acute lymphoblastic leukemia (ALL),acute myeloblastic leukemia (AML) (Seftalioglu, 2003 (a); Seftalioglu,2003 (b)), solid tissue sarcomas, colon carcinomas as well as otherhematologic malignancies and solid tumors that express CD138 (Carbone etal., 1999; Sebestyen et al., 1999; Han et al., 2004; Charnaux et al.,2004; O'Connell et al., 2004; Orosz and Kopper, 2001).

Other cancers that have been shown to be positive for CD138 expressionare many ovarian adenocarcinomas, transitional cell bladder carcinomas,kidney clear cell carcinomas, squamous cell lung carcinomas; breastcarcinomas and uterine cancers (see, for example, Davies et al., 2004;Barbareschi et al., 2003; Mennerich et al., 2004; Anttonen et al., 2001;Wijdenes, 2002).

In the normal human hematopoietic compartment, CD138 expression isrestricted to plasma cells (Wijdenes, 1996; Chilosi, 1999) and CD138 isnot expressed on peripheral blood lymphocytes, monocytes, granulocytes,and red blood cells. In particular, CD34⁺ stem and progenitor cells donot express CD138 and anti-CD138 mAbs do not affect the number of colonyforming units in hematopoietic stem cell cultures (Wijdenes, 1996). Innon-hematopoietic compartments, CD138 is mainly expressed on simple andstratified epithelia within the lung, liver, skin, kidney and gut. Onlya weak staining was seen on endothelial cells (Bernfield, 1992; Vooijs,1996). It has been reported that CD138 exists in polymorphic forms inhuman lymphoma cells (Gattei, 1999).

Monoclonal antibodies B-B4, BC/B-B4, B-B2, DL-101, 1 D4, MI15, 1.BB.210,2Q1484, 5F7, 104-9, 281-2 in particular B-B4 have been reported to bespecific to CD138. Of those B-B4, 1D4 and MI15 recognized both theintact molecule and the core protein of CD138 and were shown torecognize either the same or closely related epitopes (Gattei, 1999).Previous studies reported that B-B4 did not recognize soluble CD138, butonly CD138 in membrane bound form (Wijdenes, 2002).

B-B4, a murine IgG1 mAb, binds to a linear epitope between residues90-95 of the core protein on human syndecan-1 (CD138) (Wijdenes, 1996;Dore, 1998). Consistent with the expression pattern of CD138, B-B4 wasshown to strongly react with plasma cell line RPMI8226, but not to reactwith endothelial cells. Also consistent with the expression pattern ofCD138, B-B4 also reacted with epithelial cells lines A431 (keratinocytederived) and HepG2 (hepatocyte derived). An immunotoxin B-B4-saporin wasalso highly toxic towards the plasma cell line RPMI8226, in factconsiderably more toxic than free saporin. However, from the twoepithelial cell lines tested, B-B4-saporin showed only toxicity towardscell line A431, although in a clonogenic assay B-B4 saporin showed noinhibitory effect on the outgrowth of A431 cells (Vooijs, 1996). Otherresearchers reported lack of specificity of MM-associated antigensagainst tumors (Couturier, 1999).

An antibody/immunoconjugate “consisting essentially of” certaincomponents means in the context of the present invention that theantibody/immunoconjugate consists of the specified components and anyadditional materials or components that do not materially affect thebasic characteristics of the antibody.

The present invention uses the term “tumor cell” to include cancer cellsas well as pre-cancerous cells which may or may not form part of a solidtumor.

A “targeting agent” according to the present invention is able toassociate with a molecule expressed by a target cell and includespeptides and non-peptides. In particular, targeting agents according tothe present invention include targeting antibodies andnon-immunoglobulin targeting molecules, which may be based onnon-immunoglobulin proteins, including, but not limited to, AFFILIN®molecules, ANTICALINS® and AFFIBODIES®. Non-immunoglobulin targetingmolecules also include non-peptidic targeting molecules such astargeting DNA and RNA oligonucleotides (aptamers), but alsophysiological ligands, in particular ligands of the antigen in question,such as CD138.

A “targeting antibody” according to the present invention is or is basedon a natural antibody or is produced synthetically or by geneticengineering and binds to an antigen on a cell or cells (target cell(s))of interest. A targeting antibody according to the present inventionincludes a monoclonal antibody, a polyclonal antibody, a multispecificantibody (for example, a bispecific antibody), or an antibody fragment.The targeting antibody may be engineered to, for example, improve itsaffinity to the target cells (Ross, 2003) or diminish itsimmunogenicity. The targeting antibody may be attached to a liposomalformulation including effector molecules (Carter, 2003). An antibodyfragment comprises a portion of an intact antibody, preferably theantigen binding or variable region of the intact antibody. Examples ofantibody fragments according to the present invention include Fab, Fab′,F(ab′)₂, and Fv fragments, but also diabodies; domain antibodies (dAb)(Ward, 1989; U.S. Pat. No. 6,005,079); linear antibodies; single-chainantibody molecules; and multispecific antibodies formed from antibodyfragments. In a single chain variable fragment antibody (scFv) the heavyand light chains (VH and VL) can be linked by a short amino acid linkerhaving, for example, the sequence (glycine₄serine)_(n), which hassufficient flexibility to allow the two domains to assemble a functionalantigen binding pocket. Addition of various signal sequences may allowfor more precise targeting of the targeting antibody. Addition of thelight chain constant region (CL) may allow dimerization via disulphidebonds, giving increased stability and avidity. Variable regions forconstructing the scFv can, if a mAb against a target of interest isavailable, be obtained by RT-PCR which clones out the variable regionsfrom mRNA extracted from the parent hybridoma. Alternatively, the scFvcan be generated de novo by phage display technology (Smith, 2001). Asused herein, the term “functional fragment”, when used in reference to atargeting antibody, is intended to refer to a portion of the targetingantibody which is capable of specifically binding an antigen that isspecifically bound by the antibody reference is made to. A bispecificantibody according to the present invention may, for example, have atleast one arm that is reactive against a target tissue and one arm thatis reactive against a linker moiety (United States Patent Publication20020006379). A bispecific antibody according to the present inventionmay also bind to more than one antigen on a target cell (Carter, 2003).An antibody according to the present invention may be modified by, forexample, introducing cystein residues to introduce thiol groups(Olafsen, 2004).

In accordance with the present invention, the targeting antibody may bederived from any source and may be, but is not limited to, a camelantibody, a murine antibody, a chimeric human/mouse antibody or achimeric human/monkey antibody, in particular, a chimeric human/mouseantibody such as nBT062.

Humanized antibodies are antibodies that contain sequences derived froma human-antibody and from a non-human antibody and are also within thescope of the present invention. Suitable methods for humanizingantibodies include CDR-grafting (complementarity determining regiongrafting) (EP 0 239 400; WO 91/09967; U.S. Pat. Nos. 5,530,101; and5,585,089), veneering or resurfacing (EP 0 592 106; EP 0 519 596;Padlan, 199; Studnicka et al., 1994; Roguska et al., 1994), chainshuffling (U.S. Pat. No. 5,565,332) and Delmmunosation™ (Biovation,LTD). In CDR-grafting, the mouse complementarity-determining regions(CDRs) from, for example, mAb B-B4 are grafted into human variableframeworks, which are then joined to human constant regions, to create ahuman B-B4 antibody (hB-B4). Several antibodies humanized byCDR-grafting are now in clinical use, including MYLOTARG (Sievers etal., 2001) and HECEPTIN (Pegram et al, 1998).

The resurfacing technology uses a combination of molecular modeling,statistical analysis and mutagenesis to alter the non-CDR surfaces ofantibody variable regions to resemble the surfaces of known antibodiesof the target host. Strategies and methods for the resurfacing ofantibodies, and other methods for reducing immunogenicity of antibodieswithin a different host, are disclosed, for example, in U.S. Pat. No.5,639,641. Human antibodies can be made by a variety of methods known inthe art including phage display methods. See also U.S. Pat. Nos.4,444,887, 4,716,111, 5,545,806, and 5,814,318; and international patentapplication publications WO 98/46645, WO 98/50433, WO 98/24893, WO98/16654, WO 96/34096, WO 96/33735, and WO 91/10741.

Targeting antibodies that have undergone any non-natural modificationsuch as chimeric human/mouse antibodies or a chimeric human/monkeyantibodies, humanized antibodies or antibodies that were engineered to,for example, improve their affinity to the target cells or diminishtheir immunogenicity but also antibody fragments, in particularfunctional fragments of such targeting antibodies that have undergoneany non-natural modification, diabodies; domain antibodies; linearantibodies; single-chain antibody molecules; and multispecificantibodies are referred to herein as engineered targeting antibodies.

Chimerized antibodies, maintain the antibody binding region (ABR or Fabregion) of the non-human antibody, e.g., the murine antibody they arebased on, while any constant regions may be provided for by, e.g., ahuman antibody. Generally, chimerization and/or the exchange of constantregions of an antibody will not affect the affinity of an antibodybecause the regions of the antibody which contribute to antigen bindingare not affected by this exchange. In a preferred embodiment of thepresent invention, the engineered, in particular chimerized, antibody ofthe present invention, may have a higher binding affinity (as expressedby K_(D) values) than the respective non-human antibody it is based on.In particular, the nBT062 antibody and antibodies based thereon may havehigher antibody affinity than the murine B-B4. In another preferredembodiment of the present invention, immunoconjugates comprising thoseengineered/chimerized antibodies also display this higher antibodyaffinity. These immunconjugates may also display in certain embodimentsother advantageous properties, such as a higher reduction of tumor loadthan their B-B4 containing counterparts. In a preferred embodiment, theengineered, in particular chimerized targeting antibodies displaybinding affinities that are characterized by dissociation constantsK_(D) (nM) of less than 1.6, less than 1.5 or about or less than 1.4,while their murine counterparts are characterized by dissociationconstants K_(D) (nM) of about or more than 1.6. Immunoconjugatescomprising targeting agents such as targeting antibodies may becharacterized by dissociation constants of K_(D) (nM) of less than 2.6,less than 2.5, less than 2.4, less than 2.3, less than 2.2, less than2.1, less than 2.0, less than or about 1.9 are preferred, whileimmunoconjugates comprising the murine counterpart antibodies may becharacterized by dissociation constants K_(D) (nM) of about or more than2.6 (compare Table 3, Materials and Methods).

Fully human antibodies may also be used. Those antibodies can beselected by the phage display approach, where CD138 or an antigenicdeterminant thereof is used to selectively bind phage expressing, forexample, B-B4 variable regions (see, Krebs, 2001). This approach isadvantageously coupled with an affinity maturation technique to improvethe affinity of the antibody. All antibodies referred to herein areisolated antibodies.

In one embodiment, the targeting antibody is, in its unconjugated form,moderately or poorly internalized. Moderate internalization constitutesabout 30% to about 75% internalization of antibody, poor internalizationconstitutes about 0.01% to up to about 30% internalization after 3 hoursincubation at 37° C. In another preferred embodiment the targetingantibody binds to CD138, for example, antibodies B-B4, BC/B-B4, B-B2,DL-101, 1 D4, MI15, 1.BB.210, 2Q1484, 5F7, 104-9, 281-2 in particularB-B4. Hybridoma cells, which were generated by hybridizing SP02/0myeloma cells with spleen cells of Balb/c mice have been deposited withthe DSMZ-Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH,Mascheroder Weg 1, D-38124 Braunschweig on Dec. 11, 2007. Theidentification number of these B-B4 expressing hybridoma cells is DSMACC2874. In another embodiment, the targeting antibody does notsubstantially bind non-cell-surface expressed CD138. When, in thecontext of the present invention, the name of a specific antibody iscombined with the term “targeting antibody” such as “nBT062 targetingantibody,” this means that this targeting antibody has the bindingspecificity of the antibody nBT062. If a targeting antibody is said tobe “based on” a specified antibody, this means that this targetingantibody has the binding specificity of this antibody, but might takeany form consistent with the above description of a targeting antibody.When, in the context of the present invention, the name of a specificantigen is combined with the term “targeting antibody” such as “CD138targeting antibody,” this means that this targeting antibody has thebinding specificity for CD138. If, in the context of the presentinvention, for example, a targeting antibody is said to do something“selectively” such as “selectively targeting cell-surface expressedCD138” or, to be “selective” for something, this means that there is asignificant selectivity (i.e. a higher affinity towards CD138-positivecells compared with CD138-negative cells) for, in the case of theexample provided, cell-surface expressed CD138, compared to any otherantigens. Adverse side effects in a given environment are substantiallyreduced or even avoided due to this selectivity.

“Non-immunoglobulin targeting molecules” according to the presentinvention include targeting molecules derived from non-immunoglobulinproteins as well as non-peptidic targeting molecules. Smallnon-immunoglobulin proteins which are included in this definition aredesigned to have specific affinities towards, in particular surfaceexpressed CD138. These small non-immunoglobulin proteins includescaffold based engineered molecules such as Affilin® molecules that havea relatively low molecular weight such as between 10 kDa and 20 kDa.Appropriate scaffolds include, for example, gamma crystalline. Thosemolecules have, in their natural state, no specific binding activitytowards the target molecules. By engineering the protein surfacesthrough locally defined randomization of solvent exposed amino acids,completely new binding sites are created. Former non-binding proteinsare thereby transformed into specific binding proteins. Such moleculescan be specifically designed to bind a target, such as CD138, and allowfor specific delivery of one or more effector molecules (see, scilProteins GmbH at www.scilproteins.com, 2004). Another kind ofnon-immunoglobulin targeting molecules are derived from lipocalins, andinclude, for example ANTICALINS®, which resemble in structure somewhatimmunoglobulins. However, lipocalins are composed of a singlepolypeptide chain with 160 to 180 amino acid residues. The bindingpocket of lipocalins can be reshaped to recognize a molecule of interestwith high affinity and specificity (see, for example, Beste et al.,1999). Artificial bacterial receptors such as those marketed under thetrademark Affibody® (Affibody AB) are also within the scope of thepresent invention. These artificial bacterial receptor molecules aresmall, simple proteins and may be composed of a three-helix bundle basedon the scaffold of one of the IgG-binding domains of Protein A(Staphylococcus aureus). These molecules have binding properties similarto many immunoglobulins, but are substantially smaller, having amolecular weight often not exceeding 10 kDa and are also comparativelystable. Suitable artificial bacterial receptor molecules are, forexample, described in U.S. Pat. Nos. 5,831,012; 6,534,628 and 6,740,734.

Other “non-immunoglobulin targeting molecules” are physiological ligandsof the antigen in question. Physiological ligands of CD138 include forexample, but not limited to, ADAMTS4 (aggrecanase-1), antithrombin-3,bFGF, cathepsin G, CCL5 (RANTES), CCL7, CCL11, CCL17, CD44, collagens(collagen type 1, collagen type 2, collagen type 3, collagen type 4,collagen type 5, collagen type 6), CXCL1, elastase, gp120, HGF[hepatocyte growth factor], laminin-1, laminin-2, laminin-5, midkine,MMP-7, neutrophil elastase, and pleiotrophin (HBNF, HBGF-8).Non-peptidic targeting molecules include, but are not limited to, to DNAand RNA oligonucleotides that bind to CD138 (aptamers).

An “effector molecule” according to the present invention is a moleculeor a derivative, or an analogue thereof that is attached to a targetingagent, in particular a targeting antibody and/or an engineered targetingantibody, and that exerts a desired effect, for example, apoptosis, oranother type of cell death, or a continuous cell cycle arrest on thetarget cell or cells. Effector molecules according to the presentinvention include molecules that can exert desired effects in a targetcell and include, but are not limited to, toxins, drugs, in particularlow molecular weight cytotoxic drugs, radionuclides, biological responsemodifiers, pore-forming agents, ribonucleases, proteins of apoptoticsignaling cascades with apoptosis-inducing activities, cytotoxicenzymes, prodrug activating enzymes, antisense oligonucleotides,antibodies or cytokines as well as functional derivatives oranalogues/fragments thereof. Toxins may include bacterial toxins, suchas, but not limited to, Diphtheria toxin or Exotoxin A, plant toxins,such as but not limited to, Ricin. Proteins of apoptotic signalingcascades with apoptosis-inducing activities, include, but are notlimited to, Granzyme B, Granzyme A, Caspase-3, Caspase-7, Caspase-8,Caspase-9, truncated Bid (tBid), Bax and Bak.

In a preferred embodiment, the effector increases internal effectordelivery of the immunoconjugate, in particular when the natural form ofthe antibody on which the targeting antibody of the immunoconjugate isbased is poorly internalizable. In another preferred embodiment theeffector is, in its native form, non-selective. In certain embodimentsthe effector has high non-selective toxicity, including systemictoxicity, when in its native form. The “native form” of an effectormolecule of the present invention is an effector molecule before beingattached to the targeting agent to form an immunoconjugate. In anotherpreferred embodiment, the non-selective toxicity of the effectormolecule is substantially eliminated upon conjugation to the targetingagent. In another preferred embodiment, the effector molecule causes,upon reaching the target cell, death or continuous cell cycle arrest inthe target cell. A drug-effector molecule according to the presentinvention includes, but is not limited to, a drug including, forexample, small highly cytotoxic drugs that act as inhibitors of tubulinpolymerization such as maytansinoids, dolastatins, auristatin andcrytophycin; DNA alkylating agents like CC-1065 analogues or derivatives(U.S. Pat. Nos. 5,475,092; 5,585,499; 6,716,821) and duocarmycin;enediyne antibiotics such as calicheamicin and esperamicin; and potenttaxoid (taxane) drugs (Payne, 2003). Maytansinoids and calicheamicinsare particularly preferred. An effector maytansinoid includesmaytansinoids of any origin, including, but not limited to syntheticmaytansinol and maytansinol analogue and derivative. Doxorubicin,daunomycin, methotrexate, vinblastine, neocarzinostatin, macromycin,trenimon and α-amanitin are some other effector molecules within thescope of the present invention. Also within the scope of the presentinvention are antisense DNA molecules as effector molecules. When thename of, for example, a specific drug or class of drugs is combinedherein with the term “effector” or “effector molecule,” reference ismade to an effector of an immunoconjugate according to the presentinvention that is based on the specified drug or class of drugs.

Maytansine is a natural product originally derived from the Ethiopianshrub Maytenus serrata (Remillard, 1975; U.S. Pat. No. 3,896,111). Thisdrug inhibits tubulin polymerization, resulting in mitotic block andcell death (Remillard, 1975; Bhattacharyya, 1977; Kupchan, 1978). Thecytotoxicity of maytansine is 200-1000-fold higher than that ofanti-cancer drugs in clinical use that affect tubulin polymerization,such as Vinca alkaloids or taxol. However, clinical trials of maytansineindicated that it lacked a therapeutic window due to its high systemictoxicity. Maytansine and maytansinoids are highly cytotoxic but theirclinical use in cancer therapy has been greatly limited by their severesystemic side-effects primarily attributed to their poor selectivity fortumors. Clinical trials with maytansine showed serious adverse effectson the central nervous system and gastrointestinal system.

Maytansinoids have also been isolated from other plants including seedtissue of Trewia nudiflora (U.S. Pat. No. 4,418,064)

Certain microbes also produce maytansinoids, such as maytansinol and C-3maytansinol esters (U.S. Pat. No. 4,151,042).

The present invention is directed to maytansinoids of any origin,including synthetic maytansinol and maytansinol analogues which aredisclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870;4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268;4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348;4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,371,533; 4,424,219 and4,151,042.

In a preferred embodiment, the maytansinoid is a thiol-containingmaytansinoid and is more preferably produced according to the processesdisclosed in U.S. Pat. No. 6,333,410 to Chari et al or in Chari et al.(Chari, 1992).

DM-1 (N²-deacetyl-N²-(3-mercapto-1-oxopropyl)-maytansine) is a preferredeffector molecule in the context of the present invention. DM1 is 3- to10-fold more cytotoxic than maytansine, and has been converted into apro-drug by linking it via disulfide bond(s) to a monoclonal antibodydirected towards a tumor-associated antigen. Certain of these conjugates(sometimes called “tumor activated prodrugs” (TAPs)) are not cytotoxicin the blood compartment, since they are activated upon associating witha target cells and internalized, thereby releasing the drug (Blattler,2001). Several antibody-DM1 conjugates have been developed (Payne,2003), and been evaluated in clinical trials. For example, huC242-DM1treatment in colorectal cancer patients was well tolerated, did notinduce any detectable immune response, and had a long circulation time(Tolcher, 2003).

Other particularly preferred maytansinoids comprise a side chain thatcontains a sterically hindered thiol bond such as, but not limited to,maytansinoidsN^(2′)-deacetyl-N^(2′)-(4-mercapto-1-oxopentyl)-maytansine, alsoreferred to as “DM3,” andN^(2′)-deacetyl-N^(2′)-(4-methyl-4-mercapto-1-oxopentyl)-maytansine,also referred to as “DM4.” The synthesis of DM4 is shown in FIGS. 3 and4 and is described elsewhere herein. DM4 differs from DM1 and DM3 inthat it bears methyl groups at its αC. This results in a stericalhindrance when DM4 is attached via a linker in particular, but notlimited to, a linker comprising a disulfide bond, to a targeting agentsuch as nBT062. A wide variety of maytansinoids bearing a stericallyhindered thiol group (possessing one or two substituents, in particularalkyls substituents, such as the methyl substituents of DM4) aredisclosed U.S. Patent Publication 2004/0235840, published Nov. 25, 2004,which is incorporated herein in its entirety by reference. The sterichindrance conferred by alkyl groups such as the methyl groups on thecarbon adjacent to the sulfur atom of DM3 and DM4 may affect the rate ofintracellular cleavage of the immunoconjugate. The variable alkyl unitmay therefore affect potency, efficacy, and safety/toxicity in vitro andin vivo.

As reported by Goldmahker et al. in U.S. Patent Publication2006/0233814, such a hindrance induces alkylation (e.g., methylation) ofthe free drug, once the drug is released at its target. The alkylationmay increase the stability of the drug allowing for the so-calledbystander effect. However, as the person skilled in the art willappreciate, other effector molecules comprising substitutents such asalkyl groups at positions that result in a sterical hindrance when theeffector is attached to a targeting agent via a linker are part of thepresent invention (U.S. Patent Publication 2004/0235840). Preferablythis hindrance induces a chemical modification such as alkylation of thefree drug to increase its overall stability, which allows the drug tonot only induce cell death or continuous cell cycle arrest in CD138expressing tumor cells but, optionally, also to affect auxiliary cellsthat, e.g., support or protect the tumor from drugs, in particular cellsof the tumor stroma and the tumor vasculature and which generally do notexpress CD138 to diminish or lose their supporting or protectingfunction.

DNA alkylating agents are also particularly preferred as effectormolecules and include, but are not limited to, CC-1065 analogues orderivatives. CC-1065 is a potent antitumor-antibiotic isolated fromcultures of Streptomyces zelensis and has been shown to be exceptionallycytotoxic in vitro (U.S. Pat. No. 4,169,888). Within the scope of thepresent invention are, for example the CC-1065 analogues or derivativesdescribed in U.S. Pat. Nos. 5,475,092, 5,585,499 and 5,739,350. As theperson skilled in the art will readily appreciate, modified CC-1065analogues or derivatives as described in U.S. Pat. No. 5,846,545 andprodrugs of CC-1065 analogues or derivatives as described, for example,in U.S. Pat. No. 6,756,397 are also within the scope of the presentinvention. In certain embodiments of the invention, CC-1065 analogues orderivatives may, for example, be synthesized as described in U.S. Pat.No. 6,534,660.

Another group of compounds that make preferred effector molecules aretaxanes, especially highly potent ones and those that contain thiol ordisulfide groups. Taxanes are mitotic spindle poisons that inhibit thedepolymerization of tubulin, resulting in an increase in the rate ofmicrotubule assembly and cell death. Taxanes that are within the scopeof the present invention are, for example, disclosed in U.S. Pat. Nos.6,436,931; 6,340,701; 6,706,708 and United States Patent Publications20040087649; 20040024049 and 20030004210. Other taxanes are disclosed,for example, in U.S. Pat. No. 6,002,023, U.S. Pat. No. 5,998,656, U.S.Pat. No. 5,892,063, U.S. Pat. No. 5,763,477, U.S. Pat. No. 5,705,508,U.S. Pat. No. 5,703,247 and U.S. Pat. No. 5,367,086. As the personskilled in the art will appreciate, PEGylated taxanes such as the onesdescribed in U.S. Pat. No. 6,596,757 are also within the scope of thepresent invention.

Calicheamicin effector molecules according to the present inventioninclude gamma 1I, N-acetyl calicheamicin and other derivatives ofcalicheamicin. Calicheamicin binds in a sequence-specific manner to theminor groove of DNA, undergoes rearrangement and exposes free radicals,leading to breakage of double-stranded DNA, resulting in cell apoptosisand death. One example of a calicheamicin effector molecule that can beused in the context of the present invention is described in U.S. Pat.No. 5,053,394.

An immunoconjugate according to the present invention comprises at leastone targeting agent, in particular targeting antibody and one effectormolecule. The immunoconjugate might comprise further molecules forexample for stabilization. For immunoconjugates, the term “conjugate” isgenerally used to define the operative association of the targetingagent with one or more effector molecules and is not intended to refersolely to any type of operative association, and is particularly notlimited to chemical “conjugation”. So long as the targeting agent isable to bind to the target site and the attached effector functionssufficiently as intended, particularly when delivered to the targetsite, any mode of attachment will be suitable. The conjugation methodsaccording to the present invention include, but are not limited to,direct attachment of the effector molecule to the targeting antibody,with or without prior modification of the effector molecule and/or thetargeting antibody or attachment via linkers. Linkers can be categorizedfunctionally into, for example, acid labile, photolabile linkers, enzymecleavable linkers, such as linkers that can be cleaved by peptidases.Cleavable linkers are, in many embodiments of the invention preferred.Such cleavable linkers can be cleaved under conditions present in thecellular environment, in particular, an intracellular environment andthat have no detrimental effect on the drug released upon cleavage. LowpHs such as pH of 4 to 5 as they exist in certain intracellulardepartments will cleave acid labile linkers, while photolabile linkerscan be cleaved by, e.g., infrared light. However, linkers that arecleaved by/under physiological conditions present in the majority ofcells are preferred and are referred to herein as physiologicallycleavable linkers. Accordingly, disulfide linkers are being preferred inmany embodiments of the invention. These linkers are cleavable throughdisulfide exchange, which can occur under physiological conditions.Preferred heterobifunctional disulfide linkers include, but are notlimited to, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) (see,e.g., Carlsson et al. (1978)), N-succinimidyl4-(2-pyridyldithio)butanoate (SPDB) (see, e.g., U.S. Pat. No.4,563,304), N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP) (see,e.g., CAS Registry number 341498-08-6), N-succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (see, e.g.,Yoshitake et al., (1979)), and N-succinimidyl4-methyl-4-[2-(5-nitro-pyridyl)-dithio]pentanoate (SMNP) (see, e.g.,U.S. Pat. No. 4,563,304). The most preferred linker molecules for use inthe inventive composition are SPP, SMCC, and SPDB.

Other suitable linkers may include “non-cleavable” bonds, such as, butnot limited to Sulfosuccinimidyl maleimidomethyl cyclohexane carboxylate(SMCC), which is a heterobifunctional linker capable of linkingcompounds with SH-containing compounds. Bifunctional andheterobifunctional linker molecules, such as carbohydrate-directedheterobifunctional linker molecules, such asS-(2-thiopyridyl)-L-cysteine hydrazide (TPCH), are also within the scopeof the present invention (Vogel, 2004). The effector molecule, such as amaytansinoid, may be conjugated to the targeting antibody via a tworeaction step process, including as a first step modification of thetargeting antibody with a cross-linking reagent such as N-succinimidylpyridyldithiopropionate (SPDP) to introduce dithiopyridyl groups intothe targeting antibody. In a second step, a reactive maytansinoid havinga thiol group, such as DM1, may be added to the modified antibody,resulting in the displacement of the thiopyridyl groups in the modifiedantibody, and the production of disulfide-linked cytotoxicmaytansinoid/antibody conjugate (U.S. Pat. No. 5,208,020). However,one-step conjugation processes such as the one disclosed in UnitedStates Patent Publication 20030055226 to Chari et al are also within thescope of the present invention. In one embodiment of the presentinvention multiple effector molecules of the same or different kind areattached to a targeting antibody. As discussed elsewhere herein, thenature of the linkers employed may influence bystander killing (Kovtunet al., 2006). See also discussion of FIG. 13.

CC-1065 analogues or derivatives may be conjugated to the targetingagent via for example PEG linking groups as described in U.S. Pat. No.6,716,821.

Calicheamicins may be conjugated to the targeting antibodies via linkers(U.S. Pat. No. 5,877,296 and U.S. Pat. No. 5,773,001) or according tothe conjugation methods disclosed in U.S. Pat. No. 5,712,374 and U.S.Pat. No. 5,714,586. Another preferred method for preparing calicheamicinconjugates is disclosed in Unites States Patent Publication 20040082764.The immunoconjugates of the present invention may take the form ofrecombinant fusion proteins.

The term “cytotoxic agents” comprises “cytotoxic/cancer drugs” includingchemotherapeutic agents such as melphalan, cyclophosphamide,vincristine, doxorubicin and liposomal doxorubicin (DOXIL),cyclophosphamide, etoposide, cytarabine and cisplatin, corticosteroidssuch as prednisone and dexamethasone and agents such as thalidomide,bortezomib, lenalidomide, but also kinase inhibitor such as sorafenib orHDAC (histone deacetylase) inhibitors such as romidepsin as well asgrowth inhibitory agents, anti-hormonal agents, anti-angiogenic agents,cardioprotectants, immunostimulatory agents, immunosuppressive agents,angiogenesis inhibitors, protein tyrosine kinase (PTK) inhibitors. Alsoincluded in this definition are antibody based cytotoxic agentsincluding immunoconjugates and antibodies that have an art recognizedcytotoxic effect. Anti-CD40 is a preferred antibody. Other antibodiesinclude, but are not limited to, e.g., AVASTIN (bevacizuab) orMYELOMACIDE (milatuzumab).

THALOMID (α-(N-phthalimido) glutarimide; thaliomide), is animmunomodulatory agent. The empirical formula for thalidomide isC₁₃H₁₀N₂O₄ and the gram molecular weight is 258.2. The CAS number ofthalidomide is 50-35-1. It appears to have multiple actions, includingthe ability to inhibit the growth and survival of myeloma cells invarious ways and to inhibit the growth of new blood vessels.

VELCADE is a proteasome inhibitor used to treat multiple myeloma. It isbelieved that VELCADE acts on myeloma cells to cause cell death, and/oracts indirectly to inhibit myeloma cell growth and survival by acting onthe bone microenvironment. Without being limited to a specific theory ormode of action, VELCADE thus disrupts normal cellular processes,resulting in proteasome inhibition that promotes apoptosis.

REVLIMID is an immunomodulatory agent. It is thought that REVLIMIDaffects multiple pathways in myeloma cells, thereby inducing apoptosis,inhibiting myeloma cell growth, inhibiting vasculare entdothelial growthfactor (VEGF) thereby inhibiting angiogenesis, and reducing adhesion ofmyeloma cells to bone marrow stromal cells.

Dexamethasone is a synthetic glucocorticoid steroid hormone that acts asan anti-inflammatory and immunosuppressant. When administered to cancerpatients, dexamethasone can counteract side effects of cancer therapy.Dexamethasone can also be given alone or together with other anticanceragents, including thalidomide, adriamycin or vincristine.

The term “in combination with” is not limited to the administration atexactly the same time. Instead, the term encompassed administration ofthe immunoconjugate of the present invention and the other regime (e.g.radiotherapy) or agent, in particular the cytotoxic agents referred toabove in a sequence and within a time interval such that they may acttogether to provide a benefit that is increased compared to treatmentwith only either the immunoconjugate of the present invention or, e.g.,the other agent or agents. It is preferred that the immunoconjugate andthe other agent or agents act additively, and especially preferred thatthey act synergistically. Such molecules are suitably provided inamounts that are effective for the purpose intended. The skilled medicalpractitioner can determine empirically, or by considering thepharmacokinetics and modes of action of the agents, the appropriate doseor doses of each therapeutic agent, as well as the appropriate timingsand methods of administration. As used in the context of the presentinvention “co-administration” refers to administration at the same timeas the immunoconjugate, often in a combined dosage form.

The term “sequence identity” refers to a measure of the identity ofnucleotide sequences or amino acid sequences. In general, the sequencesare aligned so that the highest order match is obtained. “Identity”, perse, has recognized meaning in the art and can be calculated usingpublished techniques. (See, e.g.: Computational Molecular Biology, Lesk,A. M., ed., Oxford University Press, New York, 1988; Biocomputing:Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M.,and Griffin, H. G., eds., Humana Press, New Jersey, 1994; SequenceAnalysis in Molecular Biology, von Heinje, G., Academic Press, 1987; andSequence Analysis Primer, Gribskov, M. and Devereux, J., eds., MStockton Press, New York, 1991). While there exist a number of methodsto measure identity between two polynucleotide or polypeptide sequences,the term “identity” is well known to skilled artisans (Carillo, H. &Lipton, D., SIAM J Applied Math 48:1073 (1988)).

Whether any particular nucleic acid molecule is at least 50%, 60%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, forinstance, the nBT062 nucleic acid sequence, or a part thereof, can bedetermined conventionally using known computer programs such as DNAsissoftware (Hitachi Software, San Bruno, Calif.) for initial sequencealignment followed by ESEE version 3.0 DNA/protein sequence software(cabot@trog.mbb.sfu.ca) for multiple sequence alignments.

Whether the amino acid sequence is at least 50%, 60%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance SEQ IDNO:1 or SEQ ID NO:2, or a part thereof, can be determined conventionallyusing known computer programs such the BESTFIT program (WisconsinSequence Analysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711).BESTFIT uses the local homology algorithm of Smith and Waterman,Advances in Applied Mathematics 2:482-489 (1981), to find the bestsegment of homology between two sequences.

When using DNAsis, ESEE, BESTFIT or any other sequence alignment programto determine whether a particular sequence is, for instance, 95%identical to a reference sequence according to the present invention,the parameters are set such that the percentage of identity iscalculated over the full length of the reference nucleic acid or aminoacid sequence and that gaps in homology of up to 5% of the total numberof nucleotides in the reference sequence are allowed.

If, in the context of the present invention, reference is made to acertain sequence identity with a combination of residues of a particularsequence, this sequence identity relates to the sum of all the residuesspecified.

The basic antibody molecule is a bifunctional structure wherein thevariable regions bind antigen while the remaining constant regions mayelicit antigen independent responses. The major classes of antibodies,IgA, IgD, IgE, IgG and IgM, are determined by the constant regions.These classes may be further divided into subclasses (isotypes). Forexample, the IgG class has four isotypes, namely, IgG1, IgG2, IgG3, andIgG4 which are determined by the constant regions. Of the various humanantibody classes, only human IgG1, IgG2, IgG3 and IgM are known toeffectively activate the complement system. While the constant regionsdo not form the antigen binding sites, the arrangement of the constantregions and hinge region may confer segmental flexibility on themolecule which allows it to bind with the antigen.

Different IgG isotypes can bind to Fc receptors on cells such asmonocytes, B cells and NK cells, thereby activating the cells to releasecytokines. Different isotypes may also activate complement, resulting inlocal or systemic inflammation. In particular, the different IgGisotypes may bind FcγR to different degrees. FcγRs are a group ofsurface glycoproteins belonging to the Ig superfamily and expressedmostly on leucocytes. The FcγR glycoproteins are divided into threeclasses designated FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). WhileIgG1, IgG2 and IgG3 bind strongly to a variety of these classes of FcγRglycoproteins, IgG4 display much weaker binding. In particular, IgG4 isan intermediate binder of FcγRI, which results in relatively low or evenno ADCC (antibody dependent cellular cytotoxicity), and does not bind toFcγRIIIA or FcγRIIA. IgG4 is also a weak binder of FcγRIIB, which is aninhibitory receptor. Furthermore, IgG4 mediates only weak or nocomplement fixation and weak or no complement dependent cytotoxicity(CDC). In the context of the present invention, IgG4 may be specificallyemployed to prevent Fc-mediated targeting of hepatic FcR as it displaysno interaction with FcRyII on LSECs (liver sinusoidal endothelialcells), no or weak interaction with FcRγI-III on Kupffer cells(macrophages) and no interaction with FcRγIII on hepatic NK cells.Certain mutations that further reduce any CDC are also part of thepresent invention For example IgG4 residues at positions 327, 330 and331 were shown to reduce ADCC (antibody dependent cellular cytotoxicity)and CDC (Amour, 1999; Shields, 2001). One of more mutations thatstabilize the antibody are also part of the present invention (alsoreferred to herein as “stabilizing mutations”). Those mutations includein particular, leucine-to-glutamic acid mutations in the CH2 region ofIgG4 and serine-to-proline exchanges in the IgG4 hinge core. Thesemutations decrease, in certain embodiments of the invention, the amountof half-molecules to less than 10%, less than 5% and preferably lessthan 2% or 1%. Moreover, the in vivo half life of so stabilizedantibodies might be increased several days, including 1, 2, 3, 4 or morethan 5 days (Schuurman, 1999).

Targeting agents, including targeting antibodies disclosed herein mayalso be described or specified in terms of their binding affinity toantigen, in particular to CD138. Preferred binding affinities oftargeting agents such as targeting antibodies are characterized bydissociation constants K_(D) (nM) of less than 1.6, less than 1.5 orabout or less than 1.4. For immunoconjugates comprising said targetingagents such as targeting antibodies dissociation constants K_(D) (nM) ofless than 1.6, less than 1.5 ablaut or less than 2.5, less than 2.4,less than 2.3, less than 2.2, less than 2.1, less than 2.0, less than orabout 1.9 are preferred.

An antigen binding region (ABR) according to the present invention willvary based on the type of targeting antibody or engineered targetingantibody employed. In a naturally occurring antibody and in mostchimeric and humanized antibodies, the antigen binding region is made upof a light chain and the first two domains of a heavy chain. However, ina heavy chain antibody devoid of light chains, the antigen bindingregion will be made up of, e.g., the first two domains of the heavychain only, while in single chain antibodies (ScFv), which combine in asingle polypeptide chain the light and heavy chain variable domains ofan antibody molecule, the ABR is provided by only one polypeptidemolecule. FAB fragments are usually obtained by papain digestion andhave one light chain and part of a heavy chain and thus comprise an ABRwith only one antigen combining site. On the other hand, diabodies aresmall antibody fragments with two antigen-binding regions. In thecontext of the present invention, however, an antigen binding region ofan targeting antibody or engineered targeting antibody is any regionthat primarily determines the binding specificity of the targetingantibody or engineered targeting antibody.

If an ABR or another targeting antibody region is said to be “of acertain antibody”, e.g., a human or non-human antibody, this means inthe context of the present invention that the ABR is either identical toa corresponding naturally occurring ABR or is based thereon. An ABR isbased on a naturally occurring ABR if it has the binding specificity ofthe naturally occurring ABR. However, such an ABR may comprise, e.g.,point mutations, additions, deletions or posttranslational modificationsuch as glycosylation. Such an ABR may in particular have more than 70%,more than 80%, more than 90%, preferably more than 95%, more than 98% ormore than 99% sequence identity with the sequence of the naturallyoccurring ABR.

Homogenous targeting of a targeting agent such as a targeting antibody,but in particular an immunoconjugate comprising the same, in the contextof the present invention, is a measure of the variance associated withobtaining the desired result of said targeting with the targeting agent.In certain embodiments of the invention, the desired result is obtainedby simple binding to the target. This is, for example, the case inembodiments in which a certain targeting agent provides a shield againstsubsequent binding. However, the homogeneity of a targeting agent can bereadily assessed, e.g., via the efficacy of an immunoconjugatecomprising said targeting agent. For example, the efficacy of saidimmunoconjugate against a tumor antigen such as CD138 that comprises aneffector aimed at destroying tumor cells and/or arresting the growth ofa tumor can be determined by the degree of growth suppression of a tumorcomprising cells expressing the CD138 antigen. Such an immunoconjugatemay display a high variance in its efficacy. It may, for example, arresttumor growth sometimes with high efficacy, but other times with anefficacy that hardly exceeds the efficacy of the control. A low variancein the efficacy of an immunoconjugate, on the other hand, shows that theimmunoconjugate and/or targeting agent, respectively, provide thedesired result consistently. One way of quantifying the homogeneity oftargeting is to calculate the targeting variation. In the context oftumor growth arrested by an immunoconjugate comprising a certaintargeting agent, the targeting variation can be calculated by firstdetermining the time for a tumor to reach a predetermined volume, e.g.300 mm³. Preferably, the predetermined volume is chosen so that anytumor growth before and after reaching said predetermined volume issteadily increasing at about the same rate. After such time has beendetermined for a group of subjects the mean of these times (T_(m)) inthe group of subjects (e.g., SCID mice or another suitable modeldisplaying homogenous tumor growth) is calculated. T_(m) is thencorrelated to the observations made in the subject of the group showingthe least efficacy in targeting and thus being associated with tumorsthat need the least time (T_(f)) to reach said predetermined volume,and, on the other hand, the subject in the group showing the highestefficacy in targeting and thus being associated with tumors that needthe most time (T_(s)) to reach said predetermined volume by calculatingthe targeting variation for the predetermined volume according to thefollowing formula:

TARGETING VARIATION [%]=Ts−Tf/Tm×100

In a preferred embodiment, the targeting variation of theimmunoconjugate comprising the engineered targeting antibody of thepresent invention is less than 150%, less than 140%, less than 130%,less than 120%, less than 110%, less than 100%, less than 90%, less than80%, less than 70%, less than 60%, or less than 50%, and in certainembodiments even less than 45%. Preferably, the targeting variation isbetween about 10% and about 150%, preferably between about 10% and about100%, about 10% and about 80%, about 10% and about 70%, about 10% andabout 60%, about 10% and about 50%.

The homogenity of targeting can be also quantified by other means suchas determining the tumor growth delay. Also, as the person skilled inthe art will readily understand tumor volume of a certain size is onlyone parameter on which basis targeting variation may be determined.Depending on the desired result, other parameters include time (for,e.g., measuring tumor growth delay) or % of binding may be employed. Theperson skilled in the art can readily determine such other parameters.

FIG. 9 shows in (C) and (D) the differences in homogenity oftargeting/binding between immunoconjugates comprising murine antibodyBB4 (BB4-SPP-DM1; FIG. 9C) and the engineered targeting antibody nBT062(nBT062-SPP-DM1; FIG. 9D) based thereon. As can be seen from thesegraphs, results obtained with the immunoconjugate comprising theengineered targeting antibody are substantially more homogenous than theones obtained with the immunoconjugates comprising the murine antibody.This is particularly notable since the antibody binding region of BB4was not modified in nBT062. Thus, the immunoconjugate comprising theantibody binding region of the murine antibody, but no other parts ofthe murine antibody, showed properties that far exceeded results theperson skilled in the art would have expected.

nBT062 (see also FIG. 1) is a murine human chimeric IgG4 mAb achimerized version of B-B4. This chimerized version of B-B4 was createdto reduce the HAMA (Human Anti-Mouse Antibody) response, whilemaintaining the functionality of the antibody binding region of the B-B4for CD138. Surprisingly, the results obtained using an immunoconjugatecomprising the engineered targeting antibody were much more homogenous(the variance in the results was reduced). The protocol for producingnBT062 is specified below. Chinese hamster ovary cells expressing nBT062have been deposited with the DSMZ-Deutsche Sammlung von Mikroorganismenand Zellkulturen GmbH, Mascheroder Weg 1, D-38124 Braunschweig on Dec.11, 2007. The identification number is DSM ACC2875. A CD138 specificchimeric antibody based on B-B4 is generically referred to herein asc-B-B4.

The amino acid sequence for both, the heavy and the light chains hasbeen predicted from the translation of the nucleotide sequence fornBT062. The amino acid sequences predicted for the heavy chain and lightchain are presented in Table 1. Predicted variable regions are bolded,predicted CDRs are underlined.

TABLE 1  Predicted Amino Acid Sequence for nBT062nBT062 heavy chain predicted sequence (SEQ ID NO: 1): 1QVQLQQSGSE LMMPGASVKI SCKATGYTFS  NYWIE WVKQR PGHGLEWIGE 51ILPGTGRTIY NEKFKGKA TF TADISSNTVQ MQLSSLTSED SAVYYCAR RD 101YYGNFYYAMD Y WGQGTSVTV SSASTKGPSV FPLAPCSRST SESTAALGCL 151VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT 201KTYTCNVDHK PSNTKVDKRV ESKYGPPCPS CPAPEFLGGP SVFLFPPKPK 251DTLMISRTPE VTCVVVDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNS 301TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQV 351YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL 401DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQKSLSLSLG (K)The C-terminal lysine is prone to clipping and might be presentdue to incomplete clipping to a certain extent. The (K) inparentesis is not part of SEQ ID NO: 1.nBT062 light chain predicted sequence (SEQ ID NO: 2): 1DIQMTQSTSS LSASLGDRVT ISC SASQGIN   NYLN WYQQKP DGTVELLIYY 51TSTLQSGVPS RFSGSGSGTD YSLTISNLEP EDIGTYYC QQ   YSKLPRT FGG 101GTKLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV 151DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 201LSSPVTKSFN RGECTable 2. shows a comparison of the general CDR definitions of Krabat andChothia and the predicted CDRs for BT062

Kabat CDR definition nBT062 Light chain CDR1: residues 24-34CDR1: residues 24-34 CDR2: residues 50-56 CDR2: residues 50-56 CDR3:residues 89-97 CDR3: residues 89-97 Heavy chain CDR1: residues 31-35CDR1: residues 31-35 CDR2: residues 50-56 CDR2: residues 51-68 CDR3:residues 95-102 CDR3: residues 99-111 Chothia CDR definition nBT062Light chain CDR1: residues 26-32 CDR1: residues 24-34 CDR2: residues50-52 CDR2: residues 50-56 CDR3: residues 91-96 CDR3: residues 89-97Heavy chain CDR1: residues 26-32 CDR1: residues 31-35 CDR2: residues52-56 CDR2: residues 51-68 CDR3: residues 96-101 CDR3: residues 99-111

BT062 is an immunoconjugate comprising the CD138 targeting chimericantibody nBT062 that is attached via a linker, here SPDB, to thecytostatic maytansinoid derivative DM4. A chemical representation ofBT062 is provided in FIGS. 1 and 2. Immunoconjugates comprising nBT062and a maytansinoid effector molecule are often characterized in terms oftheir linker and maytansinoid effector, e.g., nBT062-SMCC-DM1, is animmunoconjugate comprising nBT062, SMCC (a “noncleavable” linkercontaining a thioester bond) and DM1 as an effector. More generically,an immunoconjugate containing nBT062 and an effector molecule may alsobe described as nBT062-linker-effector or just as nBT062-effector(nBT062N, wherein N is any effector described herein.

Reference is made herein to a unhindered counterpart (UI: unhinderedimmunoconjugate) of an immunoconjugate comprising an engineeredtargeting antibody against CD138 attached to an effector molecule via acleavable linker (CL) and is described herein as UICL, which iscontrasted to an immunoconjugate in which said effector molecule issterically hindered, and contains a cleavable linker (HICL—hinderedimmunconjugate, cleavable linker). The UICL is an immunoconjugateequivalent to the HICL comprising an engineered targeting antibody inwhich the effector molecule is, however, not sterically hindered.Examples of a pair of HICL/UICL are BT062 and nBT062-SPP-DM1. Anunhindered counterpart of such an immunoconjugate comprising anon-cleavable linker (UINCL) refers to the equivalent immunoconjugatecomprising an engineered targeting antibody in which the effectormolecule is not sterically hindered and comprises a noncleavable linker.For BT062, nBT062-SMCC-DM1 would constitute an example of such anunhindered counterpart comprising an non-cleavable linker (UNICL).

A growth of a tumor inhibiting activity (=tumor growth inhibitingactivity) of an immunoconjugate is a relative measure. It describes thetumor growth inhibiting activity of a conjugate relative to the activityof the highest performing immunoconjugate whose activity is set as 100%.For example if the activity of the highest performing immunoconjugate,say, BT062, which causes a tumor growth delay (TGD) of 32 days, is setas 100%, the activity of, e.g., nBT062-DM1, which displays a tumorgrowth delay (TGD) of 18 days is calculated as follows:

Tumor Growth Inhibiting Activity=100×(TGD_(nBT062-DM1)/TGD_(BT062)),

more generically:

Tumor Growth Inhibiting Activity=100×(TGD_(Sample)/TGD_(Reference)).

Table 3 provides suitable examples from the results depicted in FIG.11B:

TABLE 3 Tumor growth delay (TGD) and % Activity of nBT062-DMx againstMOLP-8 tumor xenografts in SCID mice based on treatment groups receivinga 450 μg/kg dose. TGD* (days) % Activity** PBS 0 0 nBT062-SMCC-DM1 18 56BT062 32 100 nBT062-SPP-DM1 13 40 *Tumor growth delay in days (TGD) asmean time in days for treatment group to reach a predetermined size (160mm³) minus the mean time for the control group to reach thispredetermined size. **Tumor Growth Inhibiting Activity = 100 ×(TGD_(Sample)/TGD_(BT062)). The activity of BT062 is defined to be 100%.

In the example provided in Table 2, BT062 provides a growth of a tumorinhibiting activity that exceeds that of its unhindered counterpart(nBT062-SPP-DM1) by 60%, and a growth of a tumor inhibiting activitythat exceeds that of its unhindered counterpart immunoconjugatecomprising a non-cleavable linker (nBT062-SMCC-DM1) by 44%.

It was previously reported that a cleavable linker in e.g.,huC242-maytansinoid immunoconjugates may provides for a so calledbystander effect. Goldmahker et al. (U.S. Patent Publication2006/0233814) also disclose that the bystander effect is particularlypronounced when the effector molecule is subject to furthermodification, in particular alkylation, upon cleavage from the targetingagent. Goldmahker et al. also showed that UICL displayed better TGD thanthe respective UINCL (see, e.g., FIG. 6 of U.S. Patent Publication2006/0233814).

However, the overall effectiveness of HICL/UICL/UINCL immunoconjugatesappear to differ from immunoconjugate to immunoconjugate and/or targetto target. For example the HICL trastuzumab-SPP-DM4 was clearlyoutperformed in its ability to reduce tumor size by the UINCLtrastuzumab-SMCC-DM1, while performance of the UICL immunoconjugatetrastuzumab-SPP-DM1 substantially resembled that of the correspondingHICL (see U.S. Patent Publication 2008/0171040 to Eberts et al.), thusrendering the results obtained a function of the immunoconjugate and thetarget.

Here, yet another relationship was found. While the HICL outperformedthe UICL and UNICL, it was also surprisingly found that an UICL in ahigh single dosage regime (250 μg/kg) actually did not show any betterresults than the UINCL. In fact, the TGD in days that was observed in anUICL in such a regime was actually lower than that of the UINCL. Thisobservation became more pronounced with an increase in dosage (450μg/kg). In sum, as shown in FIG. 11A, HICL outperformed UICL in singledose experiments as well as multiple dose experiments (not shown), to anunexpected degree. In addition, the UICL was unexpectedly outperformedby UINCL at higher dosages.

The targeting agents, in particular targeting antibodies, and/orimmunoconjugates disclosed herein can be administered by any route,including intravenously, parenterally, orally, intramuscularly,intrathecally or as an aerosol. The mode of delivery will depend on thedesired effect. A skilled artisan will readily know the best route ofadministration for a particular treatment in accordance with the presentinvention. The appropriate dosage will depend on the route ofadministration and the treatment indicated, and can readily bedetermined by a skilled artisan in view of current treatment protocols.

Pharmaceutical compositions containing an unconjugated targeting agent,the immunoconjugate of the present invention and/or any furthercytotoxic agent as active ingredients can be prepared according toconventional pharmaceutical compounding techniques. See, for example,Remington's Pharmaceutical Sciences, 17th Ed. (1985, Mack PublishingCo., Easton, Pa.). Typically, effective amounts of active ingredientswill be admixed with a pharmaceutically acceptable carrier. The carriermay take a wide variety of forms depending on the form of preparationdesired for administration, for example, intravenous, oral, parenteral,intrathecal, transdermal, or by aerosol.

For oral administration, the targeting agent and/or immunoconjugateand/or cytotoxic agent can be formulated into solid or liquidpreparations such as capsules, pills, tablets, lozenges, melts, powders,suspensions or emulsions. In preparing the compositions in oral dosageform, any of the usual pharmaceutical media may be employed, such as,for example, water, glycols, oils, alcohols, flavoring agents,preservatives, coloring agents, suspending agents, and the like in thecase of oral liquid preparations (such as, for example, suspensions,elixirs and solutions); or carriers such as starches, sugars, diluents,granulating agents, lubricants, binders, disintegrating agents and thelike in the case of oral solid preparations (such as, for example,powders, capsules and tablets). Because of their ease in administration,tablets and capsules represent the most advantageous oral dosage unitform, in which case solid pharmaceutical carriers are obviouslyemployed. If desired, tablets may be sugar-coated or enteric-coated bystandard techniques. The active agent must be stable to passage throughthe gastrointestinal tract. If necessary, suitable agents for stablepassage can be used, and may include phospholipids or lecithinderivatives described in the literature, as well as liposomes,microparticles (including microspheres and macrospheres).

For parenteral administration, the targeting agent and/or theimmunoconjugate and/or cytotoxic agent may be dissolved in apharmaceutical carrier and administered as either a solution or asuspension. Illustrative of suitable carriers are water, saline,phosphate buffer solution (PBS), dextrose solutions, fructose solutions,ethanol, or oils of animal, vegetative or synthetic origin. The carriermay also contain other ingredients, for example, preservatives,suspending agents, solubilizing agents, buffers and the like. When theunconjugated targeting agent and/or immunoconjugate and/or cytotoxicagent are being administered intracerebroventricularly or intrathecally,they may also be dissolved in cerebrospinal fluid.

Dosages administered to a subject may be specified as amount, persurface area of the subject (which include humans as well as non-humananimals). The dose may be administered to such a subject in amounts,preferably, but not exclusively from about 5 mg/m² to about 300 mg/m²,including about 20 mg/m², about 50 mg/m², about 100 mg/m², about 150mg/m², about 200 mg/m² and about 250 mg/m². The targetingagents/immunoconjugates are suitably administered at one time or over aseries of treatments. In a multiple dose regime these amounts may beadministered once a day, once a week, once every two weeks, once everythree weeks, once every four weeks, one every five weeks or once everysix weeks. Loading doses with a single high dose or, alternatively,lower doses that are administered shortly after one another followed bydosages timed at longer intervals constitute a preferred embodiment ofthe present invention. In a preferred embodiment, the timing of thedosages are adjusted for a subject so that enough time has passed priorto a second and/or any subsequent treatment so that the previous dosehas been metabolized substantially, but the amount of immunoconjugatepresent in the subject's system still inhibits, delays and/or preventsthe growth of a tumor. An exemplary “repeated single dose” regimecomprises administering an initial dose of immunoconjugate of about 200mg/m² once every three weeks. Alternatively, a high initial dose may befollowed by a biweekly maintenance dose of about 150 μg/m². However,other dosage regimens may be useful. The progress of this therapy iseasily monitored by known techniques and assays. Dosage may varydepending on whether they are administered for preventive or therapeuticpurposes, the course of any previous therapy, the patient's clinicalhistory and response to the targeting agent/immunoconjugate, and thediscretion of the attending physician.

In one embodiment, the immunoconjugate is administered with one or moreadditional cytotoxic agents such as an relevant antibody or a fragmentthereof, which is efficient in treating the same disease or anadditional comorbidity; for example a CD138 specific immunoconjugate canbe administered in combination with an antibody that recognizes anotherantigen that is overexpressed in the target type of cancer. An exampleof an antibody that can be administered in combination with a CD138specific immunoconjugate is an anti-CD40 antibody (Tai et al., 2006).

Other anti-cancer antibodies and immunoconjugates that can beco-administered with the immunoconjugates disclosed herein, includingbut not limited to, e.g., AVASTIN (bevacizuab) or MYELOMACIDE(milatuzumab).

The immunoconjugate of the present invention may also particularly beadministered in in treatment regimens with high-dose chemotherapy(preferably, melphalan, melphalan/prednisone (MP),vincristine/doxorubicin/dexamethasone (VAD), liposomaldoxorubicin/vincristine, dexamethasone (DVd), cyclophosphamide,etoposide/dexamethasone/cytarabine, cisplatin (EDAP)), stem celltransplants (e.g., autologous stem cell transplantation or allogeneicstem cell transplantation, and/or mini-allogeneic (non-myeloablative)stem cell transplantation), steroids (e.g., corticosteroids,dexamethasone, thalidomide/dexamethasone, prednisone,melphalan/prednisone), supportive therapy (e.g., bisphosphonates, growthfactors, antibiotics, intravenous immunoglobulin, low-dose radiotherapy,and/or orthopedic interventions), THALOMID (thalidomide, Celgene),VELCADE (bortezomib, Millennium), and/or REVLIMID (lenalidomide)(Chelgene Corporation) and/or other multiple myeloma treatmentsincluding radiation therapy.

If an immunoconjugate of the present invention is administered incombination with a cytotoxic agents, the above doses and regimes areoften maintained. However, if the immunconjugate and the cytotoxic agentare co-administered, low dosages of each of these therapeutic componentsare, in certain embodiments, preferred. In such a situation, theimmunconjugate may be administered at doses from about 5 mg/m² to about200 mg/m², including about 20 mg/m², about 50 mg/m², about 100 mg/m²,about 150 mg/m², while the cytotoxic agent is administered at doses thatare below the recommendation when administered alone, such at about 80%to 20% of the recommended dose.

The experimental data obtained in the cell culture based (FIG. 7) andmouse experiments (FIGS. 8 to 11), was further confirmed withexperiments that further supported these finding.

The pathogenesis of multiple myeloma involves binding of myeloma cells,via cell-surface adhesion molecules, to bone marrow stroma cells (BMSCs)as well as the extracellular matrix (ECM). This binding triggers, andthus can be made ultimately responsible, for multiple myeloma cellgrowth, drug resistance, and migration of MM cells in the bone marrowmilieu (Munshi et al. 2008). In particular, the adhesion of multiplemyeloma cells to ECM via syndecan-1 (CD138) to type I collagen, inducesthe expression of matrix metalloproteinase 1, thus promoting boneresorption and tumour invasion (Hideshima et al. 2007). Interactionsbetween multiple myeloma cells and the bone marrow microenvironmentresults in activation of a pleiotropic proliferative and anti-apoptoticcascade.

Following the homing of multiple myeloma cells to the bone marrowstromal compartment, adhesion between multiple myeloma cells and BMSCsupregulates many cytokines like interleukin-6 (IL-6) and insulin likegrowth factor 1 (IGF-1) which have angiogenic and tumor growth promotingactivities (Hideshima et al. 2007). The signalling cascades initiated bythese cytokines eventually result in MM cell resistance to conventionaltherapeutics (Anderson et al. 2000; Hideshima et al. 2006).

The in vivo efficacy of nBT062-SPDB-DM4 and nBT062-SPP-DM1 againstCD138-positive tumor cells in the presence of human bone marrow wasanalyzed in a mouse model and the results of this analysis are shown inFIG. 12. The Figure shows that both HICL and UICL perform well in thisenvironment. The increase in the level of shuIL-6R which can, in thismodel, be used as a parameter of MM cell growth, were both suppressed bythe these immunoconjugates.

In accordance with the present invention, MM is treated as follows, withthe use of BT062 as an example. This example is not intended to limitthe present invention in any manner, and a skilled artisan could readilydetermine other immunoconjugate or nBT062 based systems that are withinthe scope of the present invention and other treatment regimes whichcould be utilized for the treatment of diseases such as MM.

Due to the selective expression of CD138 on patient MM cells on via theblood stream accessible cells, the specificity of nBT062 and thestability of BT062 in the bloodstream, BT062 removes the systemictoxicity of DM4 and provides an opportunity to target the delivery ofthe DM4-effector molecule(s) homogenously. The immunoconjugates of thisinvention provide a means for the effective administration of theeffector molecules to cell sites where the effector molecules can bereleased from the immunoconjugates. This targeted delivery and releaseprovides a significant advance in the treatment of multiple myeloma, forwhich current chemotherapy methods sometimes provide incompleteremission.

In accordance with the present invention multiple myeloma is alsotreated as follows: One or more cytotoxic agents are administered in thedosages and dosage forms and according to establish treatment protocolsfor these cytotoxic agents to an individual that is also treated with animmunoconjugate of the present invention.

In particular, a patient is subjected to a treatment regime using anoral dosage of melphalan according to the manufacturer's instruction(e.g. a pill traded under the trademark ALKERAN) and an appropriatedosage of BT062, e.g., 100 mg/m² according to the present invention atcertain intervals, e.g., at the beginning or end of a melphalantreatment session, to complement the effect of the melphalan treatment.

In accordance with the present invention, in particular solid tumors mayalso be treated as follows using BT062 as an example. This example isnot intended to limit the present invention in any manner, and a skilledartisan could readily determine other immunoconjugates of the presentinvention and other treatment regimes which could be utilized for thetreatment of solid tumors. The tumor is first treated to reduce the sizeof the tumor, for example chemotherapeutically, e.g., using liposomaldoxorubicin, or radioactively. Subsequent administration of BT062 thisinvention provides a highly effective means for eliminating residualcancer cells. The administration of the immunoconjugate allows specifictargeting of these residual cells and release of the effector moleculesat the target site. The high efficacy of the immunoconjugate allows, inpreferred embodiments, for a single dose regime. This targeted deliveryand release provides a significant advance in the treatment of residualcancer cells of solid tumors, for which current chemotherapy methodssometimes provide incomplete remission.

The present invention is further described by reference to the followingExamples, which are offered by way of illustration and are not intendedto limit the invention in any manner. Standard techniques well known inthe art or the techniques specifically described below are utilized.

Materials and Methods

Chimeric Antibody Construction (cB-B4: nBT062)

B-B4

Murine antibody B-B4 as previously characterized (Wijdenes et al., Br JHaematol., 94 (1996), 318) was used in these experiments.

Cloning and Expression of B-B4 and cB-B4/nBT062

Standard recombinant DNA techniques were performed as described indetail in text books, for example in J. Sambrook; Molecular Cloning, ALaboratory Manual; 2nd Ed. (1989), Cold Spring Harbor Laboratory Press,USA, or as recommended by the manufacturer's instruction in the caseswhen kits were used. PCR-cloning and modification of the mouse variableregions have been conducted using standard PCR methodology. Primersindicated in the respective results section have been used.

Expression of cB-B4/nBT062

Exponentially growing COS cells, cultured in DMEM supplemented with 10%FCS, 580 μg/ml L-glutamine, 50 Units/ml penicillin and 50 μg/mlstreptomycin were harvested by trypsinisation and centrifugation andwashed in PBS. Cells were resuspended in PBS to a final concentration of1×10⁷ cells/ml. 700 μl of COS cell suspension was transferred to a GenePulser cuvette and mixed with heavy and kappa light chain expressionvector DNA (10 μg each or 13 μg of Supervector). Cells wereelectroporated at 1900 V, 25 μF using a Bio-Rad Gene Pulser. Transformedcells were cultured in DMEM supplemented with 10% gamma-globulin freeFBS, 580 μg/ml L-glutamine, 50 Units/ml penicillin and 50 μg/mlstreptomycin for 72 h before antibody-containing cell culturesupernatants were harvested.

Capture ELISA to Measure Expression Levels of cB-B4/nBT062

96 well plates were coated with 100 μl aliquots of 0.4 μg/ml goatanti-human IgG antibody diluted in PBS (4° C., overnight). Plates werewashed three times with 200 μl/well washing buffer (PBS+0.1% Tween-20).Wells were blocked with 0.2% BSA, 0.02% Tween-20 in PBS, before additionof 200 μl cell culture supernatants containing the secreted antibody(incubation at 37° C. for one hour). The wells were washed six timeswith washing buffer, before detection of bound antibody with goatanti-human kappa light chain peroxidase conjugate.

Purification of cB-B4/nBT062 from Cell Culture Supernatants

The cB-B4 antibody was purified from supernatants of transformed COS 7cells using the Protein A ImmunoPure Plus kit (Pierce, Rockford, Ill.),according to the manufacturer's recommendation.

cB-B4 Binding and Competition AssayAnalysis of binding activity of B-B4 and cB-B4 to CD138 was performedusing the Diaclone (Besançon, France) sCD138 kit according to themanufacturer's recommendation, considering the changes described in theresults section.RNA Preparation and cDNA Synthesis

Hybridoma B-B4 cells were grown and processed using the Qiagen Midi kit(Hilden, Germany) to isolate RNA following the manufacturer's protocol.About 5 μg of B-B4 RNA was subjected to reverse transcription to produceB-B4 cDNA using the Amersham Biosciences (Piscataway, N.J.) 1st strandsynthesis kit following the manufacturer's protocol.

Cloning of B-B4 Immunoglobulin cDNA

Immunoglobulin heavy chain (IgH) cDNA was amplified by PCR using the IgHprimer MHV7 (5′-ATGGGCATCAAGATGGAGTCACAGACCCAGG-3′) [SEQ ID NO:3] andthe IgG1 constant region primer MHCG1 (5′-CAGTGGATAGACAGATGGGGG-3′) [SEQID NO:4]. Similarly, immunoglobulin light chain (IgL) was amplifiedusing the three different IgK primers MKV2(5′-ATGGAGACAGACACACTCCTGCTATGGGTG-3′) [SEQ ID NO:5], MKV4(5′-ATGAGGGCCCCTGCTCAGTTTTTTGGCTTCTTG-3′) [SEQ ID NO:6] and MKV9(5′-ATGGTATCCACACCTCAGTTCCTTG-3′) [SEQ ID NO:7], each in combinationwith primer MKC (5′-ACTGGATGGTGGGAAGATGG-3′) [SEQ ID NO:8]. Allamplification products were directly ligated with the pCR2.1-TOPO vectorusing the TOPO-TA cloning kit (Invitrogen, Carlsbad, Calif.) accordingto the manufacturer's instruction.

E. coli TOP10 bacteria (Invitrogen) transformed with the ligated pCR2.1vector constructs were selected on LB-ampicillin-Xgal agar plates. Smallscale cultures were inoculated with single white colonies, grownovernight and plasmids were isolated using the QIAprep Spin Miniprep kitaccording to the manufacturer's instruction.

cDNA Sequence Determination

Plasmids were sequenced using the Big Dye Termination v3.0 CycleSequencing Ready Reaction Kit (ABI, Foster City, Calif.). Each selectedplasmid was sequenced in both directions using the 1210 and 1233 primerscycled on a GeneAmp9600 PCR machine. The electrophoretic sequenceanalysis was done on an ABI capillary sequencer.

The complete cycle of RT-PCR, cloning and DNA sequence analysis wasrepeated to obtain three completely independent sets of sequenceinformation for each immunoglobulin chain.

B-B4 VK DNA Sequence

1st strand synthesis was performed in three independent reactions. ThePCR products generated by using primers MKC and MKV2 (sequences givenabove) were ligated into pCR2.1-TOPO vectors according to themanufacturer's instruction. Clones from each independent set of RT-PCRreactions were sequenced in both directions. MKV2-primed productsequence was highly similar to sterile kappa transcripts originatingfrom the myeloma fusion partner such as MOPC-21, SP2 and Ag8 (Carroll etal., Mol Immunol., 25 (1988), 991; Cabilly et al., Gene, 40 (1985); 157)and was therefore disregarded.

The PCR products using MKC with MKV4 and MKV9 primers were similar toeach other and differed only at the wobble positions within the leadersequence primer.

B-B4 VH DNA Sequence

1st strand synthesis was performed in three independent reactions andPCR products were cloned and sequenced from each 1st strand product.Five clones were sequenced from each 1st strand.

Construction of Chimeric cB-B4 Expression Vectors

The construction of the chimeric expression vectors entails adding asuitable leader sequence to VH and VK, preceded by a BamHI restrictionsite and a Kozak sequence. The Kozak consensus sequence is crucial forthe efficient translation of a variable region sequence. It defines thecorrect AUG codon from which a ribosome can commence translation, andthe single most critical base is the adenine (or less preferably, aguanine) at position −3, upstream of the AUG start. The leader sequenceis selected as the most similar sequence in the Kabat database (Kabat etal., NIH National Technical Information Service, 1991). These additionsare encoded within the forward (For) primers (both having the sequence5′-AGAGAAGCTTGCCGCCACCATGATT-GCCTCTGCTCAGTTCCTTGGTCTCC-3′ [SEQ ID NO:9];restriction site is underlined; Kozak sequence is in bold type).Furthermore, the construction of the chimeric expression vectors entailsintroducing a 5′ fragment of the human gamma1 constant region, up to anatural ApaI restriction site, contiguous with the 3′ end of the Jregion of B-B4 and, for the light chain, adding a splice donor site andHindIII site. The splice donor sequence is important for the correctin-frame attachment of the variable region to its appropriate constantregion, thus splicing out the V:C intron. The kappa intron+CK areencoded in the expression construct downstream of the B-B4 VK sequence.Similarly, the gamma-4 CH is encoded in the expression constructdownstream of the B-B4 VH sequence.

The B-B4 VH and Vκ genes were first carefully analyzed to identify anyunwanted splice donor sites, splice acceptor sites, Kozak sequences andfor the presence of any extra sub-cloning restriction sites which wouldlater interfere with the subcloning and/or expression of functionalwhole antibody. An unwanted HindIII site was found in the Vκ sequencewhich necessarily was removed by site-directed mutagenesis via PCRwithout changing the amino acid sequence. For this reactions,oligonucleotide primers BT03 (5′-CAACAGTATAGTAAGCTCCCTCGGACGTTCGGTGG-3′)[SEQ ID NO:10] and BT04 (5′-CCACCGAACGTCCGAGGGAGCTTACTATACTGTTG-3′) [SEQID NO:11] were used and mutagenesis was performed according to theStratagene (La Jolla, Calif.) Quickchange Mutagenesis Kit protocol.

Kappa Chain Chimerization Primers

The non-ambiguous B-B4 Vκ leader sequence, independent of the PCR primersequence, was aligned with murine leader sequences in the Kabatdatabase. The nearest match for the B-B4 VH leader was VK-10 ARS-A (Sanzet al., PNAS, 84 (1987), 1085). This leader sequence is predicted to becut correctly by the SignalP algorithm (Nielsen et al., Protein Eng, 10(1997); 1). Primers CBB4Kfor (see above) and g2258(5′-CGCGGGATCCACTCACGTTTGATTTCCAGCTTGGTGCCTCC-3′ [SEQ ID NO:12];Restriction site is underlined) were designed to generate a PCR productcontaining this complete leader, the B-B4 Vκ region, and HindIII andBamHI terminal restriction sites, for cloning into the pKN100 expressionvector. The forward primer, CBB4K introduces a HindIII restriction site,a Kozak translation initiation site and the VK-10 ARS-A leader sequence.The reverse primer g2258 introduces a splice donor site and a BamHIrestriction site. The resulting fragment was cloned into theHindIII/BamHI restriction sites of pKN100.

Heavy Chain Chimerization Primers

The non-ambiguous B-B4 VH leader sequence, independent of the PCR primersequence, was aligned with murine leader sequences in the Kabatdatabase. The nearest match for the B-B4 VK leader was VH17-1A (Sun etal, PNAS, 84 (1987), 214). This leader sequence is predicted to be cutcorrectly by the SignalP algorithm. Primers cBB4Hfor (see above) andg22949 (5′-CGATGGGCCCTTGGTGGAGGCTGAGGA-GACGGTGACTGAGGTTCC-3′ [SEQ IDNO:13]; Restriction site is underlined) were designed to generate a PCRproduct containing VH17-1A leader, the B-B4 VH region, and terminalHindIII and ApaI restriction sites, for cloning into the pG4D200expression vector. The forward primer cBBHFor introduces a HindIIIrestriction site, a Kozak translation initiation site and the VH17-1Aleader sequence. The reverse primer g22949 introduces the 5′ end of thegamma4 C region and a natural ApaI restriction site. The resultingfragment was cloned into the HindIII/ApaI restriction sites of pG4D200,resulting in vector pG4D200cBB4.

Production of cBB4 Antibody

One vial of COS 7 cells was thawed and grown in DMEM supplemented with10% Fetal clone I serum with antibiotics. One week later, cells (0.7 mlat 10⁷ cells/nil) were electroporated with pG4D200cBB4 plus pKN100cBB4(10 μg DNA each) or no DNA. The cells were plated in 8 ml growth mediumfor 4 days. Electroporation was repeated seven times.

Detection of Chimeric Antibody

A sandwich ELISA was used to measure antibody concentrations in COS 7supernatants. Transiently transformed COS 7 cells secreted about 6956ng/ml antibody (data not shown).

Binding Activity of cB-B4

To assay the binding activity of cB-B4 in COS 7 culture supernatants,the Diaclone sCD138 kit has been used, a solid phase sandwich ELISA. Amonoclonal antibody specific for sCD138 has been coated onto the wellsof the microtiter strips provided. During the first incubation, sCD138and biotinylated B-B4 (bio-B-B4) antibody are simultaneously incubatedtogether with a dilution series of unlabeled test antibody (B-B4 orcB-B4).

The concentrations of bio-B-B4 in this assay have been reduced in orderto obtain competition with low concentrations of unlabeled antibody(concentration of cB-B4 in COS 7 cell culture supernatants wereotherwise too low to obtain sufficient competition). Results from thisassay reveal that both antibodies have the same specificity for CD138(data not shown).

Purification of cB-B4

Chimeric B-B4 was purified from COS 7 cell supernatants using theProtein A ImmunoPure Plus kit (Pierce), according to the manufacturer'srecommendation (data not shown).

K_(D)-Determination: Comparison nBT062/BB4

Purification of soluble CD138 Soluble CD138 antigen from U-266 cellculture supernatant was purified by FPLC using a 1 mL “HiTrapNHS-activated HP” column coupled with B-B4. Cell culture supernatant wasloaded in PBS-Buffer pH 7.4 onto the column and later on CD138 antigenwas eluted with 50 mM tri-ethylamine pH 11 in 2 mL fractions. ElutedCD138 was immediately neutralised with 375 μL 1 M Tris-HCl, pH 3 toprevent structural and/or functional damages.

Biotinylation of CD138

Sulfo-NHS-LC (Pierce) was used to label CD138. NHS-activated biotinsreact efficiently with primary amino groups like lysine residues in pH7-9 buffers to form stable amide bonds.

For biotinylation of CD138, 50 μl of CD138 were desalted using proteindesalting spin columns (Pierce). The biotinylation reagent (EZ-LinkSulfo NHS-LC-Biotin, Pierce) was dissolved in ice-cooled deionised H₂Oto a final concentration of 0.5 mg/mL. Biotinylation reagent and capturereagent solution were mixed having a 12 times molar excess ofbiotinylation reagent compared to capture reagent (50 pmol CD138 to 600pmol biotinylation reagent) and incubated 1 h at room temperature whileshaking the vial gently. The unbound biotinylation reagent was removedusing protein desalting columns.Immobilization of bCD138The sensorchip (SENSOR CHIP SA, BIACORE AB) used in the BIACORE assay isdesigned to bind biotinylated molecules for interaction analysis inBIACORE systems. The surface consists of a carboxymethylated dextranmatrix pre-immobilized with streptavidin and ready for high-affinitycapture of biotinylated ligands. Immobilization of bCD138 was performedon SENSOR CHIP SA using a flow rate of 10 μL/min by manual injection.The chip surface was conditioned with three consecutive 1-minuteinjections of 1 M NaCl in 50 mM NaOH. Then biotinylated CD138 wasinjected for 1 minute.

K_(D)-Determination of Different Antibodies Using BIACORE

The software of BIACORE C uses pre-defined masks, so called “Wizards”for different experiments where only certain settings can be changed. Asthe BIACORE C was originally developed to measure concentrations, thereis no wizard designed to carry out affinity measurements. However, withthe adequate settings, the wizard for “non-specific binding” could beused to measure affinity rate constants and was therefore used forK_(D)-determination. With this wizard, two flow cells were measured andthe dissociation phase was set to 90 s by performing the “Regeneration1” with BIACORE running buffer. “Regeneration 2” which is equivalent tothe real regeneration was performed with 10 mM Glycine-HCl pH 2.5. Afterthis step, the ligand CD138 was in its binding competent state again.During the whole procedure HBS-EP was used as running and dilutionbuffer. To determine binding of the different antibodies (˜150 kDa) toCD138, association and dissociation was analysed at differentconcentrations (100, 50, 25 12.5, 6.25 and 3.13 nM). The dissociationequilibrium constants were determined by calculating the rate constantska and kd. Afterwards, the K_(D)-values of the analytes were calculatedby the quotient of kd and ka with the BIAevaluation software. Theresults are shown in Table 4.

TABLE 4 Comparative analysis of K_(D) values of nBT062 and B- B4.Standard deviations are given for mean K_(D) values. Affinity AntibodyK_(D) (nM) mean K_(D) (nM) nBT062 1.4 1.4 +/− 0.06 1.4 1.5 B-B4 1.7 1.6+/− 0.06 1.7 1.6 nBT062-SPDB-DM4 1.9 1.9 +/− 0.00 1.9 1.9 B-B4-SPP-DM12.6 2.6 +/− 0.06 2.7 2.6

Discussion

Mean K_(D) values for each antibody were calculated from threeindependent experiments. The results show that in all measurementsnBT062 exhibits slightly decreased K_(D) values compared to B-B4 (meanK_(D) values were 1.4 and 1.6 nM, respectively).

Preparation of Immunoconjugates

nBT062-DM1 and huC242-DM1

The thiol-containing maytansinoid DM1 was synthesized from the microbialfermentation product ansamitocin P-3, as previously described by Chari(Chari et al., Cancer Res. 1 (1992), 127). Preparation of humanized C242(huC242) (Roguska et al., PNAS, 91 (1994), 969) has been previouslydescribed. Antibody-drug conjugates were prepared as previouslydescribed (Liu et al., PNAS, 93 (1996), 8618). An average of 3.5 DM1molecules was linked per antibody molecule.

nBT062-DM4

BT062 is an antibody-drug conjugate composed of the cytotoxicmaytansinoid drug, DM4, linked via disulfide bonds through a linker tothe nBT062 chimerized monoclonal antibody. Maytansinoids areanti-mitotics that inhibit tubulin polymerization and microtubuleassembly (Remillard et al., Science 189 (1977), 1002). Chemical andschematic representations of BT062 (nBT062-DM4) are shown in FIGS. 1 and2.

Synthesis of DM4

DM4 is prepared from the well known derivative maytansinol (Kupchan etal., J. Med. Chem., 21 (1978), 31). Maytansinol is prepared by reductivecleavage of the ester moiety of the microbial fermentation product,ansamitocin P3, with lithium trimethoxyaluminum hydride (see FIG. 3).

DM4 is synthesized by acylation of maytansinol withN-methyl-N-(4-methydithiopentanoyl)-L-alanine (DM4 side chain) in thepresence of dicyclohexylcarbodiimide (DCC) and zinc chloride to give thedisulfide-containing maytansinoid DM4-SMe. The DM4-SMe is reduced withdithiothreitol (DTT) to give the desired thiol-containing maytansinoidDM4 (see FIG. 4 for the DM4 process flow diagram).

Immunoconjugate BT062

The procedure for the preparation of nBT062-DM4 is outlined in FIG. 5.The nBT062 antibody is modified with N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB linker) to introduce dithiopyridyl groups. DM4 is mixedwith the modified antibody at a concentration in excess of theequivalents of dithiopyridyl groups. The BT062 conjugate forms by adisulfide exchange reaction between the thiol group of DM4 and thedithiopyridyl groups introduced into the antibody via the linker.Purification by chromatography and diafiltration removes the lowmolecular weight reactants (DM4) and reaction products (thiopyridine),as well as aggregates of conjugated antibody, to produce the bulk drugsubstance.

FACS Analysis and WST Cytotoxicity Assays FACS Analysis

OPM-2 cells are plasma cell leukemia cell lines showing highlyexpressing CD138. OPM-2 cells were incubated with nBT062,nBT062-SPDB-DM4, nBT062-SPP-DM1 or nBT062-SMCC-DM1 at differentconcentrations (indicated in FIG. 6). The cells were washed andCD138-bound antibody or conjugates were detected using afluorescence-labeled secondary antibody in FACS analysis. The meanfluorescence measured in these experiments was plotted against theantibody concentration.

Cell Viability Assay

CD138⁺ MOLP-8 cells were seeded in flat bottom plates at 3000cells/well. CD138⁻ BJAB control cells were seeded at 1000 cells/well.The cells were treated with nBT062-SPDB-DM4, nBT062-SPP-DM1 ornBT062-SMCC-DM1 at different concentrations (indicated in FIG. 7) forfive days. WST reagent (water-soluble tetrazolium salt, ROCHE) was addedin order to measure cell viability according to the manufacturer'sinstruction (ROCHE). The reagent was incubated for 7.5 h on MOLP-8 cellsand for 2 h on BJAB cells. The fraction of surviving cells wascalculated based on the optical densities measured in a microplatereader using standard procedures.

Discussion

Binding of nBT062-SPDB-DM4, nBT062-SPP-DM1, nBT062-SMCC-DM1 or nBT062was analyzed by FACS. CD138⁺ OPM-2 as target cells were incubated withnBT062 or immunoconjugates and cell-bound molecules were detected usinga fluorescence-labeled secondary antibody. In FIG. 6, the meanfluorescences as measure for the amount of cell bound antibody isplotted against different antibody or conjugate concentrations. Theresults show, that nBT062-SPDB-DM4, nBT062-SPP-DM1 and nBT062-SMCC-DM1show very similar binding characteristics. In addition, the resultsstrongly suggest that the binding characteristics of the unconjugatedantibody is not affected by the conjugated toxins.

In cell viability assays, the cytotoxic activity of the antibody againstCD138⁺ MOLP-8 target cells and against CD138⁻ BJAB B-lymphoblastomacontrol cells were analyzed. Both cell lines were seeded in flat-bottomplates and incubated with increasing concentrations of theimmunoconjugates. Unconjugated antibody was used as a control. Thecytotoxic activity was analyzed five days after addition of theimmunoconjugates by using WST reagent in order to measure cellviability. In FIG. 7 (A)-(C), the fraction of surviving cells relativeto control cells treated with vehicle control is plotted againstincreasing immunoconjugate concentrations. The results show thatcytotoxic activity of nBT062-SPDB-DM4, nBT062-SPP-DM1 andnBT062-SMCC-DM1 against MOLP-8 cells is very similar. As expected,CD138⁻ BJAB control cells were not killed by the immunoconjugates,indicating that all immunoconjugates act via cell specific binding toCD138. In competition experiments, in which MOLP-8 cells werepreincubated with a molar excess of unconjugated nBT062. Preincubationsubstantially blocked the cytotoxicity of nBT062-SPDB-DM4, providingfurther evidence that the immunoconjugates kill the cells via specificbinding to CD138 onto the cell surface (FIG. 7 (D)).

Xenograft Mouse Experiments

To evaluate the importance of CD138 targeting on the anti-tumor activityof antibody-maytansinoid conjugates of a human chimeric version of theB-B4 antibody, nBT062, xenograft mouse experiments were performed. Twoversions of nBT062-maytansinoid conjugates were prepared that may differin the chemical stability of their disulfide linkages (nBT062-SPP-DM1and nBT062-SPDB-DM4). The anti-tumor activity of these antibody-drugconjugates was compared to the activity of the B-B4-SPP-DM1 conjugate(comprising the murine parental antibody), as well as unconjugated freemaytansinoid (DM4), native unmodified nBT062 antibody, and anon-targeting (irrelevant) IgG1-maytansinoid conjugate. The conjugateswere evaluated in a CD138-positive xenograft model (MOLP-8) of humanmultiple myeloma in severe combined immunodeficient (SCID) mice.

In these mice, subcutaneous tumors were established (female CB.17 SCIDmice) by inoculation with MOLP-8 cell suspensions. Treatment with asingle bolus intravenous injection was conducted when tumor volumesreached an average 113 mm³. Changes in tumor volume and body weight weremonitored twice per week. Experiments were carried out over 68 daysafter tumor cell inoculation.

Xenograft Mouse Experiments A Mice

Female CB.17 SCID mice, five weeks old, were obtained from Charles RiverLaboratories.

Human Tumor Cell Lines

MOLP-8, a human multiple myeloma cell line, was supplied from ATCC.MOLP-8 cells, which express the CD138 antigen on their cell surface anddevelop xenograft tumors in SCID mice, were maintained in RPMI-1640medium supplemented with 4 mM L-glutamine (Biowhittaker, Walkersville,Md.), 10% fetal bovine serum (Hyclone, Logan, Utah) and 1%streptomycin/penicillin, at 37° C. in a humidified atmosphere thatcontained 5% CO₂.

Part I Tumor Growth in Mice

Each mouse was inoculated with 1×10⁷ MOLP-8 cells subcutaneously intothe area under the right shoulder. The total volume was 0.2 ml permouse, in which the ratio of serum-free medium to matrigel (BDBioscience, Bedford, Mass.) was 1/1 (v/v). Prior to treatment, thexenograft tumors were monitored daily and were allowed to becomeestablished. The tumor volume reached approximately 113 mm³ about 11days after tumor cell inoculation. Tumor take rate of CB.17 SCID micewas 100%.

Eleven days after tumor cell inoculation, 42 mice were selected based ontumor volumes and body weights. The tumor volume was in a range of 68.2to 135.9 mm³. The forty-two mice were randomly divided into seven groups(A-G) of six animals each based on tumor volume.

Each of six mice in Group A received 200 μl of PBS as vehicle control.Each mouse in group B received 13.8 mg/kg of nBT062 naked antibody. Thisdose is equivalent to the amount of nBT062 antibody component in 250μg/kg of linked maytansinoid. The ratio of molecular weights ofmaytansinoids to nBT062 antibody in a conjugate molecule is approximate1/55. Each mouse in Group C received 250 μg/kg of DM4. Each mouse inGroup D received 250 μg/kg of huC242-DM4. Mice in groups E, F and Greceived 250 μg/kg of nBT062-SPDB-DM4, B-B4-SPP-DM1 and nBT062-SPP-DM1each, respectively.

All agents were intravenously administered as a single bolus injectionthrough a lateral tail vein with a 1 ml syringe fitted with a 27 gauge,½ inch needle. Prior to administration, the stock solutions of nBT062antibody, nBT062-SPDB-DM4 and nBT062-SPP-DM1 were diluted with sterilePBS to concentrations of 2 mg/ml, 28.1 μg/ml and 28.1 μg/ml,respectively, so that the injected volume for each mouse was between120-220 μl.

Part II

In a second set of experiments, MOLP-8 cells (1.5×10⁷ cells per mouse),suspended in a 50:50 mixture of serum free media and matrigel wereinjected subcutaneously in the area under the right shoulder in 100 μl.Tumor volumes reached about 80 mm³ at day 11 and the mean of thecontrols was about 750 mm³ at day 25, post cell inoculation. The tumordoubling time was estimated to be 4.58 days. Each mouse in the controlgroup (n=6) received 0.2 ml of sterile PBS administered into the lateraltail vein (i.v.) in a bolus injection. All treatment doses were based onconjugated maytansinoid. Nine groups (n=6) were treated with a singleintravenous injection of nBT062-SMCC-DM1, nBT062-SPDB-DM4, ornBT062-SPP-DM1, each at doses of 450, 250 and 100 μg/kg. An additionalgroup (n=6) received 250 μg/kg nBT062-SMCC-DM1 in a repeated dosing(weekly for five weeks). Mice were randomized into eleven groups (n=6)by tumor volume using the LabCat Program. The tumor volumes ranged from40.0 to 152.5 mm³. The mice were dosed based on the individual bodyweight.

Tumor size was measured twice per week in three dimensions using theLabCat System (Tumor Measurement and Tracking, Innovative ProgrammingAssociated, Inc., Princeton, N.J.). The tumor volume in mm³ wascalculated using the methodology described in Tomayko et al., CancerChemother. Pharmacol, 24 (1989), 148:

Volume=Length×Width×Height×½

Log₁₀ cell kill was calculated with the formula described in Bissery etal., Cancer Res., 51 (1991), 4845:

Log₁₀ cell kill=(T−C)/T _(d)×3.32

where (T−C) or tumor growth delay, is the median time in days requiredfor the treatment group (T) and the control group (C) tumors, to reach apredetermined size (600 mm³). T_(d) is the tumor doubling time, based onthe median tumor volume in the control mice, and 3.32 is the number ofcell doublings per log of cell growth.

Results

The tumor growth in individual mice is shown in FIGS. 8 and 9. The mean(+/−SD) tumor growth for each group is shown in FIG. 10.

As compared with tumor growth in the PBS-treated animals, treatment withnBT062 antibody, unconjugated free DM4 or the irrelevant non-targetingconjugate huC242-DM4 did not cause any significant inhibition of tumorgrowth.

All three CD138-targeting conjugates, nBT062-SPDB-DM4, B-B4-SPP-DM1 andnBT062-SPP-DM1, at a dose of 250 μg/kg caused marked delay in tumorgrowth. Based on the mean tumor volumes measured in the treatmentgroups, the DM4 conjugate nBT062-SPDB-DM4 was the most active one, whilethe nBT062-SPP-DM1 conjugate showed slightly increased activity ascompared to its murine counterpart B-B4-SPP-DM1 (FIG. 10). The resultsobtained in individual mice show in addition that the anti-tumoractivity obtained with B-B4-SPP-DM1 is more heterogeneously andtherefore less predicable than that measure in mice treated withnBT062-SPP-DM1. In terms of homogeneity of anti tumor activity, theother conjugate that uses nBT062 as targeting antibody nBT062-SPDB-DM4behaved similar to nBT062-SPP-DM1.

No body weight reduction was observed in any treatment group suggestingthat the treatments were well tolerated.

Discussion

The results of the analysis of three CD138-targeting conjugates inexperimental animals demonstrate the importance of targeted delivery forthe anti-tumor activity. While the maytansinoid conjugates of the humanchimeric nBT062 and the murine B-B4 antibodies show significant activityas measured by log cell kill, there was no significant impact on tumorgrowth from treatment with unconjugated DM4, unmodified native huBT062antibody, or a non-targeting control conjugate (huC242-DM4).

The immunoconjugate prepared from the human chimeric antibody,nBT062-SPP-DM1, gave slightly higher anti-tumor activity then theconjugate prepared from its murine counterpart, B-B4-SPP-DM1. Inaddition, treatment with nBT062-SPP-DM1 and nBT062-SPDB-DM4 resulted inmore homogenous responses in individual mice as compared to treatmentwith B-B4-SPP-DM1. The high binding variation of B-B4-SPP-DM1 explainedthat the measurement of the median tumor volume (+/−SD) of MOLP-8 humanmultiple myeloma xenografts in CB.17 SCID mice over time (days)post-inoculation actually provided for relatively better results forB-B4-SPP-DM1 than for nBT062-SPP-DM1 (data not shown). This feature ofimmunoconjugates using nBT062 as a targeting antibody seems to bebeneficial especially for therapeutic use of the conjugates.

Lastly, the most potent of the maytansinoid conjugates, following singleiv administration in the MOLP-8 xenograft models in SCID mice, wasnBT062-SPDB-DM4.

Bystander Killing (Cell Viability Assay)

CD138⁺ OPM2 cells and CD138⁻ Namalwa cells were seeded in round bottomplates either in separate wells or in coculture. The cells were treatedwith nBT062-SPDB-DM4 at concentrations ranging from 1×10⁻⁸ to 1×10⁻⁹ M.The fraction of viable cells was detected using WST reagent(water-soluble tetrazolium salt, ROCHE) according to the manufacturer'sinstruction (ROCHE). The fraction of surviving cells was calculatedbased on the optical densities measured in a microplate reader usingstandard procedures.

Discussion

Bystander killing of non-target cells in close proximity (as present inround bottom wells) to multiple myeloma cells upon nBT062-SPDB-DM4treatment was analysed in an in vitro study in which CD138-positive OPM2cells were cultured in coculture with CD138-negative Namawla cells (FIG.13). Generally, while CD138-positive cells are efficiently killed bynBT062-SPDB-DM4, CD138-negative cells were not affected by theconjugate. In the coculture in round bottom wells, however,nBT062-SPDB-DM4 also killed the antigen-negative cells in closeproximity to the antigen-positive cells (an effect that is oftenreferred to as bystander killing). Kovtun et al. (2006) discussed thatbystander killing mediated by maytansinoid conjugates occurs only inclose proximity to antigen-positive cells. Kovtun et al. (2006), whichis incorporated herein by reference in its entirety, also discusses theimportance of the linker of the immunoconjugate. In vivo, bystanderkilling may contribute to 1) the eradication of tumour cells thatheterogeneously express CD138, 2) the destruction of the tumourmicroenvironment by the killing of tumour stroma cells, and 3) theprevention of the selection of CD138-negative nBT062-SPDB-DM4-resistantcells.

The bystander effect is of particular importance if the activity of animmunoconjugate is impaired by a target antigen that is expressed intumors in a heterogeneous fashion. If this is the case, a particularcell of a tumor expresses, if at all, the antigen not in amount thatwould allow effective direct targeting and killing of said cell by therespective immunoconjugate. The anti-tumor efficacy of nBT062-SPDB-DM4on CD138-negative cells in coculture with CD138-positive cells clarifiedthat the presence of target cells influences, under the appropriatecircumstances, the cytotoxic activity of nBT062-SPDB-DM4 towardsnon-target cells.

Xenograft Mouse Experiments B

In this set of experiments, eighty-five mice were inoculated with MOLP-8cells (1.5×10⁷ cells/mouse) subcutaneously in the right shoulder. Tumortake rate was 100%. Sixty-six SCID mice bearing bulky MOLP-8 tumors witha mean tumor volume of about 80 mm³ were randomized into eleventreatment groups (n=6). Mice were treated with a single dose of one ofthree conjugates (nBT062-SMCC-DM1, nBT062-SPDB-DM4 or nBT062-SPP-DM1).An additional group received five weekly doses of nBT062-SMCC-DM1 and acontrol group received a single dose of PBS. Mean tumor volumes areshown in FIG. 11A. A dose response was established for each conjugate. Amedian tumor volume of 750 mm³ in the PBS-treated animals was reached onday 25. Tumor doubling time determined by the best-fit linear regressioncurve fit on a log-linear plot of control tumor growth was 4.58 days.Animals treated with nBT062-SPDB-DM4 at 450 μg/kg had the highest logcell kill (LCK=2.89), followed by animals treated with nBT062-SMCC-DM1at 250 μg/kg weekly dosing (LCK=2.1; see Table 5). Comparison of themean tumor growth curves for the treatment groups by repeated measuresANOVA performing Dunnett's Multiple Comparison Test showed a significantdifference between the PBS control group and 450 μg/kg nBT062-SPDB-DM4(p<0.01), 250 μg/kg nBT062-SPDB-DM4 (p<0.05) and five weekly doses of250 μg/kg nBT062-SMCC-DM1 (p<0.05). No partial or complete tumorregression in any of the treatment groups occurred with the exception ofone animal receiving 450 μg/kg nBT062-SPDB-DM4, which had partialregression of the tumor until day 85 post-inoculation.

TABLE 5 Log cell kill (LCK) values as measure for anti-tumor activity ofdifferent nBT062-DMx conjugates in different dosing schemes. Refer tothe Materials and methods section for information on calculation of LCKvalues. Test Material Dose (μg/kg) LCK Dosing PBS single dosenBT062-SMCC-DM1 450 0.85 single dose nBT062-SMCC-DM1 250 0.53 singledose nBT062-SMCC-DM1 100 0 single dose nBT062-SPDB-DM4 450 2.89 singledose nBT062-SPDB-DM4 250 1.05 single dose nBT062-SPDB-DM4 100 0.39single dose nBT062-SPP-DM1 450 0.8 single dose nBT062-SPP-DM1 250 0.39single dose nBT062-SPP-DM1 100 0.2 single dose nBT062-SMCC-DM1 250 2.1weekly for 5 weeksIn Vivo Efficacy of nBT062-SPDB-DM4 and nBT062-SPP-DM1 in the BoneMarrow Environment

Preparation of SCID Mice Having Human Fetal Bone Implants

Human fetal long bones (human fetal bone chips) were implanted into theupper body of CB17 SCID-mice (SCID-hu) as previously described (Urashimaet al., 1997) and thus provided for a model in mouse for the homing ofhuman MM cells to human BM cells.

Treatment Regime (SCID-Hu/INA-6 Mice)

4 weeks following bone implantation, 2.5×10⁶ INA-6 cells in a finalvolume of 100 μL RPMI-1640 cell culture medium were injected directlyinto the human bone marrow cavity in the SCID-hu mice described above.An increase in the levels of soluble human IL-6 receptor (shuIL-6R),which is released by INA-6 cells, was used as a parameter of MM cellgrowth and disease burden.Mice developed measurable serum shuIL-6R approximately 4 weeks followingINA-6 cell injection and then received 0.176 mg conjugate or vehiclecontrol via tail vein injection weekly for 7 weeks. After eachtreatment, blood samples were collected and measured for shuIL-6R levelsby an enzyme-linked immunosorbent assay (ELISA, R&D Systems,Minneapolis, Minn.). The results are depicted in FIG. 12.

Discussion

Interleukin 6 (IL-6) is a growth and survival factor for multiplemyeloma cells. INA-6 is an IL-6-dependent human myeloma cell line, whichalso requires bone marrow stromal cells (BMSC) to proliferate. INA-6cell lines produce soluble IL-6 receptor (shuIL-6R). An increase in thelevels of shuIL-6R can be used as a parameter of MM cell growth anddisease burden.

Thus, the sCID-hu/INA-6 mice provide a model for multiple myeloma cellsgrowing in their normal bone marrow environment. The tumor cells of thismodel, which directly interact with the human bone marrow, closelyresemble the situation in patients, in which tumor cell growth is alsopromoted by the presence of stromal cells. As INA-6 cells releasesoluble human interleukin-6 receptor (shuIL-6R), serum concentrations ofthis protein can be used as a measure for tumor cell load in these mice.The in vivo potency of nBT062-SPDB-DM4 and nBT062-SPP-DM1 were tested inthis environment.

Treatment of SCIDhu/INA-6 mice with weekly i.v. administrations ofnBT062-SPDB-DM4 or nBT062-SPP-DM1 for seven weeks induced efficienttumour regression, as detected by a decrease in serum shuIL-6R levelsrelative to the control, indicating good efficacy of the conjugates evenin the environment of human bone marrow, which reflect the relevantsituation in patients (FIG. 12).

It will be appreciated that the methods and compositions of the instantinvention can be incorporated in the form of a variety of embodiments,only a few of which are disclosed herein. It will be apparent to theartisan that other embodiments exist and do not depart from the spiritof the invention. Thus, the described embodiments are illustrative andshould not be construed as restrictive.

BIBLIOGRAPHY

-   Akkina R K, Rosenblatt J D, Campbell A G, Chen I S, Zack J A.    Modeling human lymphoid precursor cell gene therapy in the SCID-hu    mouse. Blood. 1994; 84:1393-1398.-   Armour K L, Clark M R, Hadley A G, et al. Recombinant human IgG    molecules lacking Fcgamma receptor I binding and monocyte triggering    activities. Eur J Immunol. 1999; 29(8):2613-24.-   Anderson K C, Kyle R A, Dalton W S, Landowski T, Shain K, Jove R,    Hazlehurst L, Berenson J. Multiple Myeloma: New Insights and    Therapeutic Approaches. Hematology 2000; 147-165.-   Anttonen A, Heikkila P, Kajanti M, Jalkanen M, Joensuu H. High    syndecan-1 expression is associated with favourable outcome in    squamous cell lung carcinoma treated with radical surgery. Lung    Cancer. 2001 June; 32(3):297-305.-   Barbareschi M, Maisonneuve P, Aldovini D, Cangi M G, Pecciarini L,    Angelo Mauri F, Veronese S, Caffo O, Lucenti A, Palma P D,    Galligioni E, Doglioni C. High syndecan-1 expression in breast    carcinoma is related to an aggressive phenotype and to poorer    prognosis. Cancer. 2003 August 1; 98(3):474-83.-   Bataille R, Jégo G, Robillard N, Barillé-Nion S, Harousseau J L,    Moreau P, Amiot M, Pellat-Deceunynck C. The phenotype of normal,    reactive and malignant plasma cells. Identification of “many and    multiple myelomas” and of new targets for myeloma therapy.    Haematologica. 2006 September; 91(9):1234-40. Review.-   Bernfield M, Kokenyesi R, Kato M, Hinkes M T, Spring J, Gallo R L,    Lose E J. Biology of the syndecans: a family of transmembrane    heparan sulfate proteoglycans. Annu Rev Cell Biol. 1992; 8:365-393.-   Beste G, Schmidt F S, Stibora T, Skerra A. Small antibody-like    proteins with prescribed ligand specificities derived from the    lipocalin fold. Proc. Natl. Acad. Sci. USA. 1999: 96, 1898-1903.-   Bhattacharyya B, Wolff J. Maytansine binding to the vinblastine    sites of tubulin. FEBS Lett. 1977; 75:159-162.-   Bisping G, Kropff M, Wenning D, Dreyer B, Bessonov S, Hilberg F,    Roth G J, Munzert G, Stefanic M, Stelljes M, Scheffold C,    Müller-Tidow C, Liebisch P, Lang N, Tchinda J, Serve H L, Mesters R    M, Berdel W E, Kienast J. Targeting receptor kinases by a novel    indolinone derivative in multiple myeloma: abrogation of    stroma-derived interleukin-6 secretion and induction of apoptosis in    cytogenetically defined subgroups. Blood. 2006 March 1;    107(5):2079-89. Epub 2005 November 8.-   Blättler W A and Chari R V J. Drugs to Enhance the Therapeutic    Potency of Anticancer Antibodies: Antibody-Drug Conjugates as    Tumor-Activated Prodrugs. In: Ojima, I., Vite, G. D. and Altmann,    K.-H., Editors, 2001. Anticancer Agents—Frontiers in Cancer    Chemotherapy, American Chemical Society, Washington, D.C., pp.    317-338.-   Bross P F, Beitz J, Chen G, Chen X H, Duffy E, Kieffer L, Roy S,    Sridhara R, Rahman A, Williams G, Pazdur R. Approval summary:    gemtuzumab ozogamicin in relapsed acute myeloid leukemia. Clin    Cancer Res. 2001; 7:1490-1496.-   Carbone A, Gaidano G, Gloghini A, Ferlito A, Rinaldo A, Stein H.    AIDS-related plasma-blastic lymphomas of the oral cavity and jaws: a    diagnostic dilemma. Ann. Otol. Rhinol. Laryngol. 1999; 108: 95-99.-   Carlsson J, Drevin H, Axen R. Protein thiolation and reversible    protein-protein conjugation.    N-succinimidyl-3-(2-pyridyldithio)propionate, a new    heterobifunctional reagent. Biochem J 1978; 173: 723-737.-   Carter P. Improving the efficacy of antibody-based cancer therapies.    Nat Rev Cancer. 2001; 1:118-129.-   Chari R V, Martell B A, Gross J L, Cook S B, Shah S A, Blattler W A,    McKenzie S J, Goldmacher V S. Immunoconjugates containing novel    maytansinoids: promising anticancer drugs. Cancer Res. 1992;    52:127-131.-   Chari R V, Jackel K A, Bourret L A, Derr S M, Tadayoni B M, Mattocks    K M, Shah S A, Liu C, Blather W A and Goldmacher V S. Enhancement of    the selectivity and antitumor efficacy of a CC-1065 analogue through    immunoconjugate formation. Cancer Res. 1995; 55: 4079-4084.-   Charnaux N, Brule S, Chaigneau T, Saffar L, Sutton A, Hamon M, Prost    C, Lievre N, Vita C, Gattegno L. RANTES (CCL5) induces a    CCR5-dependent accelerated shedding of syndecan-1 (CD138) and    syndecan-4 from HeLa cells and forms complexes with the shed    ectodomains of these proteoglycans as well as with those of CD44.    Glycobiology. 2004 September 8 [Epub ahead of print]-   Chen B P, Galy A, Kyoizumi S, Namikawa R, Scarborough J, Webb S,    Ford B, Cen D Z, Chen S C. Engraftment of human hematopoietic    precursor cells with secondary transfer potential in SCID-hu mice.    Blood. 1994; 84:2497-2505.-   Chilosi M, Adami F, Lestani M, Montagna L, Cimarosto L, Semenzato G,    Pizzolo G, Menestrina F. CD138/syndecan-1: a useful    immunohistochemical marker of normal and neoplastic plasma cells on    routine trephine bone marrow biopsies. Mod Pathol. 1999;    12:1101-1106.-   Clement C, Vooijs, W. C., Klein, B., and Wijdenes, J. In: al. SFSe,    ed. J. Leukocyte Typing V. Oxford: Oxford University Press;    1995:714-715.-   Couturier O, Faivre-Chauvet A; Filippovich I V; Thedréz P,    Sai-Maurel C; Bardiés M; Mishra A K; Gauvrit M; Blain G; Apostolidis    C; Molinet R; Abbe J C; Bateille R; Wijdenes J; Chatal J F; Cherel    M; Validation of 213Bi-alpha radioimmunotherapy for multiple    myeloma. Clinical Cancer Research 5(10 Suppl.) (October 1999)    3165s-3170s.-   Davies E J et al., Blackhall F H, Shanks J H, David G, McGown A T,    Swindell R, Slade R J, Martin-Hirsch P, Gallagher J T, Jayson G C.    Distribution and Clinical Significance of Heparan Sulfate    Proteoglycans in Ovarian Cancer Clin Cancer Res. 2004;    10(15):5178-86.-   Dhodapkar M V, Abe E, Theus A, Lacy M, Langford J K, Barlogie B,    Sanderson R D. Syndecan-1 is a multifunctional regulator of myeloma    pathobiology: control of tumor cell survival, growth, and bone cell    differentiation. Blood. 1998; 91:2679-2688.-   Dore J M, Morard F, Vita N, Wijdenes J. Identification and location    on syndecan-1 core protein of the epitopes of B-B2 and B-B4    monoclonal antibodies. FEBS Lett. 1998; 426:67-70.-   Dowell J A, Korth-Bradley J, Liu H, King S P, Berger M S.    Pharmacokinetics of gemtuzumab ozogamicin, an antibody-targeted    chemotherapy agent for the treatment of patients with acute myeloid    leukemia in first relapse. J Clin Pharmacol. 2001; 41:1206-1214.-   Edinger M, Sweeney T J, Tucker A A, Olomu A B, Negrin R S, Contag    C H. Noninvasive assessment of tumor cell proliferation in animal    models. Neoplasia. 1999; 1:303-310.-   Gattei V, Godeas C, Degan M, Rossi F M, Aldinucci D, Pinto A.    Characterization of Anti-CD138 monoclonal antibodies as tools for    investigating the molecular polymorphism of syndecan-1 in human    lymphoma cells. Br J Haematol. 1999; 104:152-162.-   Hamann P R, Hinman L M, Beyer C F, Lindh D, Upeslacis J, Flowers D    A, Bernstein I. An anti-CD33 antibody-calicheamicin conjugate for    treatment of acute myeloid leukemia. Choice of linker. Bioconjug    Chem. 2002; 13:40-46.-   Han I, Park H, Oh E S. New insights into syndecan-2 expression and    tumourigenic activity in colon carcinoma cells. J Mol Histol. 2004:    35(3):319-26.-   Hideshima T, Catley L, Yasui H, Ishitsuka K, Raje N, Mitsiades C,    Podar K, Munshi N C, Chauhan D, Richardson P G, Anderson K C.    Perifosine, an oral bioactive novel alkylphospholipid, inhibits Akt    and induces in vitro and in vivo cytotoxicity in human multiple    myeloma cells. Blood 2006; 107(10):4053-62.-   Hideshima T, Mitsiades C, Tonon G, Richardson P G, Anderson K C.    Understanding multiple myeloma pathogenesis in the bone marrow to    identify new therapeutic targets. Nat Rev Cancer 2007; 7(8):585-98.-   Horvathova M, Gaillard, J.-P., Liutard, J., Duperray, C.,    Lavabre-Bertrand, T., Bourquard, P et al. In: al. SFSe, ed.    Leucocyte Typing V. Oxford: Oxford University Press; 1995:713-714.-   Kovtun Y V, Audette C A, Ye Y, Xie H, Ruberti M F, Phinney S J, et    al. Antibody-drug conjugates designed to eradicate tumors with    homogeneous and heterogeneous expression of the target antigen.    Cancer Res 2006; 66 (6):3214-21.-   Krebs B, Rauchenberger R, Reiffert S, Rothe C, Tesar M, Thomassen E,    Cao M, Dreier T, Fischer D, Floss A et al. High-throughput    generation and engineering of recombinant human antibodies. 2001. J.    Immunol. Methods 254, pp. 67-84.-   Kupchan S M, Sneden A T, Branfman A R, Howie G A, Rebhun L I, Mclvor    W E, Wang R W, Schnaitman T C. Structural requirements for    antileukemic activity among the naturally occurring and    semisynthetic maytansinoids. J Med Chem. 1978; 21:31-37.-   Kyoizumi S, Baum C M, Kaneshima H, McCune J M, Yee E J, Namikawa R.    Implantation and maintenance of functional human bone marrow in    SCID-hu mice. Blood. 1992; 79:1704-1711.-   Kyoizumi S, Murray L J, Namikawa R. Preclinical analysis of cytokine    therapy in the SCID-hu mouse. Blood. 1993; 81:1479-1488.-   Langford J K, Stanley M J, Cao D, Sanderson R D. Multiple heparan    sulfate chains are required for optimal syndecan-1 function. J Biol    Chem. 1998 November 6; 273(45):29965-71.-   Liu C, Tadayoni B M, Bourret L A, Mattocks K M, Derr S M, Widdison W    C, Kedersha N L, Ariniello P D, Goldmacher V S, Lambert J M,    Blattler W A, Chari R V. Eradication of large colon tumor xenografts    by targeted delivery of maytansinoids. Proc Natl Acad Sci USA. 1996;    93:8618-8623.-   McCune J M, Namikawa R, Kaneshima H, Shultz L D, Lieberman M,    Weissman I L. The SCID-hu mouse: murine model for the analysis of    human hematolymphoid differentiation and function. Science. 1988;    241:1632-1639.-   Mennerich D, Vogel A, Klaman I, Dahl E, Lichtner R B, Rosenthal A,    Pohlenz H D, Thierauch K H, Sommer A. Shift of syndecan-1 expression    from epithelial to stromal cells during progression of solid    tumours. Eur J Cancer. 2004 June; 40(9):1373-82.-   Mosmann T. Rapid colorimetric assay for cellular growth and    survival: application to proliferation and cytotoxicity assays. J    Immunol Methods. 1983; 65:55-63.-   Munshi N C, Longo D L, Anderson K C. Plasma cell disorders. In:    Braunwald E, Fauci A S, Kasper D L, Hauser S L, Longo D L, Jameson J    L, editors. Harrison's Principles of Internal Medicine. 16th ed. New    York: McGraw-Hill Medical Publishing Division; 2008. p. 700-7.-   Namikawa R, Ueda R, Kyoizumi S. Growth of human myeloid leukemias in    the human marrow environment of SCID-hu mice. Blood. 1993;    82:2526-2536.-   O'Connell F P, Pinkus J L, Pinkus G S. CD138 (Syndecan-1), a Plasma    Cell Marker Immunohistochemical Profile in Hematopoietic and    Nonhematopoietic Neoplasms. Am J Clin Pathol 2004; 121:254-263.-   Ojima I, Geng X, Wu X, Qu C, Borella C P, Xie H, Wilhelm S D, Leece    B A, Bartle L M, Goldmacher V S and Chari R V. Tumor-specific novel    taxoid-monoclonal antibody conjugates. 2002. J. Med. Chem. 45, pp.    5620-5623.-   Olafsen, T, Cheung, C C, Yazaki, P J, Li L, Sundaresan G, Gambhir S    S, Sherman, M A, Williams, L E, Shively, J E, Raubitschek, A A, and    Wu, A M. Covalent disulfide-linked anti-CEA diabody allows    site-specific conjugation and radiolabeling for tumor targeting    applications. 2004; Prot. Eng. Design & Selection 17:1: 21-27.-   Orosz Z, Kopper L. Syndecan-1 expression in different soft tissue    tumours. Anticancer Res. 2001: 21(1B):733-7.-   Padlan, E A. A possible procedure for reducing the immunogenicity of    antibody variable domains while preserving their ligand-binding    properties. Mol. Immunol. 1991; 28: 489-498.-   Payne G. Progress in immunoconjugate cancer therapeutics. Cancer    Cell. 2003; 3:207-212.-   Pegram M D, Lipton A, Hayes D F, Weber B L, Baselga J M, Tripathy D,    Baly D, Baughman S A, Twaddell T, Glaspy J A and Slamon D J. Phase    II study of receptor-enhanced chemosensitivity using recombinant    humanized anti-p185HER2/neu monoclonal antibody plus cisplatin in    patients with HER2/neu-overexpressing metastatic breast cancer    refractory to chemotherapy treatment. 1998. J. Clin. Oncol. 16, pp.    2659-2671.-   Rawstron A C, Owen R G, Davies F E, Johnson R J, Jones R A, Richards    S J, Evans P A, Child J A, Smith G M, Jack A S, Morgan G J.    Circulating plasma cells in multiple myeloma: characterization and    correlation with disease stage. Br J Haematol. 1997; 97:46-55.-   Remillard S, Rebhun L I, Howie G A, Kupchan S M. Antimitotic    activity of the potent tumor inhibitor maytansine. Science. 1975;    189:1002-1005.-   Roguska M A, Pedersen J T, Keddy C A, Henry A H, Searle S J, Lambert    J M, Goldmacher V S, Blattler W A, Rees A R, Guild B C. Humanization    of murine monoclonal antibodies through variable domain resurfacing.    Proc Natl Acad Sci USA. 1994; 91:969-973.-   Ross S, Spencer S D, Holcomb I, Tan C, Hongo J, Devaux B, Rangell L,    Keller G A, Schow P, Steeves R M, Lutz R J, Frantz G, Hillan K,    Peale F, Tobin P, Eberhard D, Rubin M A, Lasky L A, Koeppen H.    Prostate stem cell antigen as therapy target: tissue expression and    in vivo efficacy of an immunoconjugate. Cancer Res. 2002 May 1;    62(9):2546-53.-   Ross J S, Gray K, Gray G, Worland P J, Rolfe M. Anticancer    Antibodies, Am J Clin Path. (Apr. 4, 2003).-   Sanderson R D, Lalor P, Bernfield M. B lymphocytes express and lose    syndecan at specific stages of differentiation. Cell Regul. 1989;    1:27-35.-   Sandhu J S, Clark B R, Boynton E L, Atkins H, Messner H, Keating A,    Hozumi N. Human hematopoiesis in SCID mice implanted with human    adult cancellous bone. Blood. 1996; 88:1973-1982.-   Sasaki A, Boyce B F, Story B, Wright K R, Chapman M, Boyce R, Mundy    G R, Yoneda T. Bisphosphonate risedronate reduces metastatic human    breast cancer burden in bone in nude mice. Cancer Res. 1995;    55:3551-3557.-   Schneider U, van Lessen A, Huhn D, Serke S. Two subsets of    peripheral blood plasma cells defined by differential expression of    CD45 antigen. Br J Haematol. 1997; 97:56-64.-   Schuurman J, Van Ree R, G. J. Perdok G J, Van Doorn H R, Tan K Y,    Aalberse R C, Normal human immunoglobulin G4 is bispecific: it has    two different antigen-combining sites, Immunology 1999; 97:693-698.-   Sebestyen A, Berczi L, Mihalik R, Paku S, Matolcsy A, Kopper L.    Syndecan-1 (CD138) expression in human non-Hodgkin lymphomas. Br J    Haematol. 1999; 104(2):412-9.-   Seftalioglu A, Karakus S. Syndecan-1/CD138 expression in normal    myeloid, acute lymphoblastic and myeloblastic leukemia cells. Acta    Histochem. 2003; 105:213-221.-   Seftalioglu A, Karakus S, Dundar S, Can B, Erdemli E, Irmak M K,    Oztas E, Korkmaz C, Yazar F, Cavusoglu I. Syndecan-1 (CD138)    expression in acute myeloblastic leukemia cells—an immuno electron    microscopic study. Acta Oncol. 2003; 42:71-74.-   Senter P D, Doronina S, Cerveny C, Chace D, Francisco J, Klussman K,    Mendelsohn B, Meyer D, Siegall C B, Thompson J et al. (2002). Cures    and regressions of established tumors with monoclonal antibody    auristatin conjugates. Abstract #2062, American Association for    Cancer Res. (San Francisco, Calif.: American Association for Cancer    Res.), 414.-   Shields R L, Namenuk A K, Hong K, Meng Y G, Rae J, Briggs J, Xie D,    Lai J, Stadlen A, Li B, Fox J A, Presta L G. High resolution mapping    of the binding site on human IgG1 for Fc gamma RI, Fc gamma RII, Fc    gamma RIII, and FcRn and design of IgG1 variants with improved    binding to the Fc gamma R. J Biol Chem. 2001; 276(9):6591-604.-   Sievers E L, Larson R. A., Stadtmauer, E. A., Estey, E., Lowenberg,    B., Dombret, H., Karanes, C., Theobald, M., Bennett, J. M.,    Sherman, M. L. et al. Efficacy and safety of gemtuzumab ozogamicin    in patients with CD33-positive acute myeloid leukemia in first    relapse. 2001. J. Clin. Oncol. 19, pp. 3244-3254.-   Sievers E L and Linenberger M. Mylotarg: antibody-targeted    chemotherapy comes of age. 2001. Curr. Opin. Oncol. 13, pp. 522-527.-   Smith R., Single chain antibody variable region fragments; available    at the Standford website as of May, 2001.-   Studnicka G M, Soares S, Better M, Williams R E, Nadell R, Horwitz    A H. Human-engineered monoclonal antibodies retain full specific    binding activity by preserving non-CDR complementarity-modulating    residues. Protein Eng. 1994: 7(6): 805-814.-   Tai Y T, Li X F, Catley L, Coffey R, Breitkreutz I, Bae J, Song W,    Podar K, Hideshima T, Chauhan D, Schlossman R, Richardson P, Treon S    P, Grewal I S, Munshi N C, Anderson K C. Immunomodulatory drug    lenalidomide (CC-5013, IMiD3) augments anti-CD40 SGN-40-induced    cytotoxicity in human multiple myeloma: clinical implications.    Cancer Res. 2005 December 15; 65(24):11712-20.-   Tassone P, Goldmacher V S, Neri P, Gozzini A, Shammas M A, Whiteman    K A, Hylander-Gans L L, Carrasco D R, Hideshima T, Shringarpure R,    Shi J, Allam C K, Wijdenes J, Venuta S, Munshi N C, Anderson K C,    Cytotoxic activity of the maytansinoid immunoconjugate B-B4-DM1    against CD138⁺ multiple myeloma cells, Blood, 2004, 104 (12), pp.    3688-3696.-   Tolcher A W, Ochoa L, Hammond L A, Patnaik A, Edwards T, Takimoto C,    Smith L, de Bono J, Schwartz G, Mays T, Jonak Z L, Johnson R,    DeWitte M, Martino H, Audette C, Maes K, Chari R V, Lambert J M,    Rowinsky E K. Cantuzumab mertansine, a maytansinoid immunoconjugate    directed to the CanAg antigen: a phase I, pharmacokinetic, and    biologic correlative study. J Clin Oncol. 2003; 21:211-222.-   Urashima M, Chen B P, Chen S, Pinkus G S, Bronson R T, Dedera D A,    Hoshi Y, Teoh G, Ogata A, Treon S P, Chauhan D, Anderson K C. The    development of a model for the homing of multiple myeloma cells to    human bone marrow. Blood. 1997; 90:754-765.-   Vogel C W. Preparation of immunoconjugates using antibody    oligosaccharide moieties. Methods in Molecular Biology:    Bioconjugation protocols strategies and methods. 2004; 283:087-108.-   Vooijs W C, Post J, Wijdenes J, Schuurman H J, Bolognesi A, Polito    L, Stirpe F, Bast E J, de Gast G C. Efficacy and toxicity of    plasma-cell-reactive monoclonal antibodies B-B2 and B-B4 and their    immunotoxins. Cancer Immunol Immunother. 1996; 42:319-328.-   Ward, E. S., D. Gussow, A. D. Griffiths, P. T. Jones, and G. Winter.    Binding activities of a repertoire of single immunoglobin variable    domains secreted from Escherichia coli. Nature. 1989. 341:544-546.-   Wargalla U C, Reisfeld R A. Rate of internalization of an    immunotoxin correlates with cytotoxic activity against human tumor    cells. Proc. Natl. Acad. Sci. USA. 1989; 86:5146-5150.-   Wijdenes J, Vooijs W C, Clement C, Post J, Morard F, Vita N, Laurent    P, Sun R X, Klein B, Dore J M. A plasmocyte selective monoclonal    antibody (B-B4) recognizes syndecan-1. Br J Haematol. 1996;    94:318-323.-   Wijdenes J, Dore J M, Clement C, Vermot-Desroches C. CD138, J Biol    Regul Homeost Agents. 2002 April-June; 16(2):152-5.-   Witzig T E, Kimlinger T K, Ahmann G J, Katzmann J A, Greipp P R.    Detection of myeloma cells in the peripheral blood by flow    cytometry. Cytometry. 1996; 26:113-120.-   Xie H, Audette C, Hoffee M, Lambert J M, Blättler W.    Pharmacokinetics and biodistribution of the antitumor    immunoconjugate, cantuzumab mertansine (huC242-DM1), and its two    components in mice. J Pharmacol Exp Ther. 2004 March;    308(3):1073-82.-   Yang M, Jiang P, An Z, Baranov E, Li L, Hasegawa S, Al-Tuwaijri M,    Chishima T, Shimada H, Moossa A R, Hoffman R M. Genetically    fluorescent melanoma bone and organ metastasis models. Clin Cancer    Res. 1999; 5:3549-3559.-   Yang M, Baranov E, Jiang P, Sun F X, Li X M, Li L, Hasegawa S,    Bouvet M, Al-Tuwaijri M, Chishima T, Shimada H, Moossa A R, Penman    S, Hoffman R M. Whole-body optical imaging of green fluorescent    protein-expressing tumors and metastases. Proc Natl Acad Sci USA.    2000; 97:1206-1211.-   Yang Y, MacLeod V, Dai Y, Khotskaya-Sample Y, Shriver Z,    Venkataraman G, Sasisekharan R, Naggi A, Torri G, Casu B, Vlodaysky    I, Suva L J, Epstein J, Yaccoby S, Shaughnessy J D Jr, Barlogie B,    Sanderson R D. The syndecan-1 heparan sulfate proteoglycan is a    viable target for myeloma therapy. Blood. 2007 September 15;    110(6):2041-8. Epub 2007 May 29.-   Yoshitake S, Yamada Y, Ishikawa E, Masseyeff R. Conjugation of    glucose oxidase from Aspergillus niger and rabbit antibodies using    N-hydroxysuccinimide ester of    N-(4-carboxycyclohexylmethyl)-maleimide. Eur J Biochem 1979;    101:395-399.

1.-15. (canceled)
 16. A method of treating multiple myeloma in asubject, comprising: providing a immunoconjugate comprising a targetingantibody and an effector molecule, wherein the effector is linked to thetargeting antibody via a cleavable linker, and wherein saidimmunoconjugate target CD138 expressing cells, wherein said targetingantibody comprises: heavy chain variable region CDR3 comprising aminoacid residues 99 to 111 of SEQ ID NO: 1, and light chain variable regionCDR3 comprising amino acid residues 89 to 97 of SEQ ID NO: 2,respectively, and heavy chain variable region CDR1 and CDR2 comprisingamino acid residues 31 to 35 and 51 to 68 of SEQ ID NO: 1, and lightchain variable region CDR1 and CDR 2 comprising amino acid residues 24to 34 and 50 to 56 of SEQ ID NO: 2, respectively and administering tosaid subject said immunoconjugate in an amount effective to treatmultiple myeloma.
 17. The method of claim 16, wherein said cleavablelinker comprises a disulfite bond.
 18. A method for immunoconjugatemediated drug delivery comprising: providing a immunoconjugatecomprising a targeting antibody and an effector molecule, wherein theeffector is linked to the targeting antibody via a cleavable linker, andwherein said immunoconjugate targets CD138 expressing cells, whereinsaid targeting antibody comprises: heavy chain variable region CDR3comprising amino acid residues 99 to 111 of SEQ ID NO: 1, and lightchain variable region CDR3 comprising amino acid residues 89 to 97 ofSEQ ID NO: 2, respectively, and heavy chain variable region CDR1 andCDR2 comprising amino acid residues 31 to 35 and 51 to 68 of SEQ ID NO:1, and light chain variable region CDR1 and CDR 2 comprising amino acidresidues 24 to 34 and 50 to 56 of SEQ ID NO: 2, respectively of claim15, and administering said immunoconjugate in a therapeuticallyeffective amount, wherein said IgG4 isotype alleviates ADCC, complementdependent cytotoxicity and/or Fc-mediated targeting of hepatic FcR. 19.(canceled)
 20. The method of claim 18, wherein said cleavable linkercomprises a disulfite bond.
 21. The method of claim 16, wherein thecleavable linker is a SPDB (N-succinimidyl-4-(2-pyridyldithio) butyrate)linker.
 22. The method of claim 18, wherein the cleavable linker is aSPDB (N-succinimidyl-4-(2-pyridyldithio) butyrate) linker. 23.(canceled)
 24. (canceled)
 25. A method for inhibiting, delaying and/orpreventing the growth of a tumor comprising CD138 tumor cells and/orspread of tumor cells of such a tumor in a patient in need thereof,comprising administering to said patient at least one immunoconjugatecomprising a targeting antibody and an effector molecule, wherein theeffector is linked to the targeting antibody via a cleavable linker, andwherein said immunoconjugate targets CD138 expressing cells, whereinsaid targeting antibody comprises: heavy chain variable region CDR3comprising amino acid residues 99 to 111 of SEQ ID NO: 1, and lightchain variable region CDR3 comprising amino acid residues 89 to 97 ofSEQ ID NO: 2, respectively, and heavy chain variable region CDR1 andCDR2 comprising amino acid residues 31 to 35 and 51 to 68 of SEQ ID NO:1, and light chain variable region CDR1 and CDR 2 comprising amino acidresidues 24 to 34 and 50 to 56 of SEQ ID NO: 2, respectively in a growthof said tumor and/or spreading of said tumor cells inhibiting orreducing amount, wherein said immunoconjugate inhibits, delays orprevents the growth and/or spread of said tumor cells.
 26. The method ofclaim 25, wherein said patient suffers from a hematologic malignancyand/or a solid tumor comprising CD138 expressing cells.
 27. The methodof claim 26, wherein said patient suffers from one of the following:multiple myeloma, ovarian carcinoma, kidney carcinoma, gall bladdercarcinoma, breast carcinoma, prostate cancer, lung cancer, coloncarcinoma, Hodgkin's and non-Hodgkin's lymphoma, chronic lymphocyticleukemia (CLL), acute lymphoblastic leukemia (ALL), acute myeloblasticleukemia (AML), solid tissue sarcoma or colon carcinoma.
 28. The methodof claim 27, wherein the disease is multiple myeloma.
 29. The method ofclaim 25, wherein said effector molecule of said immunoconjugate is atoxin, cytotoxic enzyme, low molecular weight cytotoxic drug, apore-forming agent, biological response modifier, prodrug activatingenzyme, an antibody, cytokine or a radionuclide.
 30. The method of claim25, wherein said immunoconjugate is administered in a single dose of 5mg/m² to about 300 mg/m².
 31. The method of claim 25, wherein saidimmunoconjugate is administered in at least two doses of about 5 mg/m²to about 300 mg/m², optionally at hourly, daily, weekly intervals orcombinations thereof.
 32. A method for inhibiting, delaying and/orpreventing the growth of a tumor and/or spread of malignant tumor cellsin a patient in need thereof, comprising (a) administering to saidpatient at least one immunoconjugate comprising a targeting antibody andan effector molecule, wherein the effector is linked to the targetingantibody via cleavable linker, and wherein said immunoconjugate targetsCD138 expressing cells, wherein said targeting antibody comprises: heavychain variable region CDR3 comprising amino acid residues 99 to 111 ofSEQ ID NO: 1, and light chain variable region CDR3 comprising amino acidresidues 89 to 97 of SEQ ID NO: 2, respectively, and heavy chainvariable region CDR1 and CDR2 comprising amino acid residues 31 to 35and 51 to 68 of SEQ ID NO: 1, and light chain variable region CDR1 andCDR 2 comprising amino acid residues 24 to 34 and 50 to 56 of SEQ ID NO:2, respectively in a growth of a tumor and/or spreading of tumor cellsinhibiting, delaying or preventing amount; and (b) administering to saidpatient one or more cytotoxic agent and/or radiation in an amounteffective to reduce tumor load, wherein said immunoconjugate inhibits,delays or prevents the growth and/or spread of tumor cells comprisingCD138 expressing cells.
 33. The method of claim 32, wherein (a) and (b)are performed consecutively in two consecutive treatment regimes. 34.The method of claim 32, wherein the immunoconjugate of (a) and the atleast one cytotoxic agent of (b) are co-administered.
 35. The method ofclaim 32, wherein the cytotoxic agent is mephalan, vincristine,doxorubicin, dexamethasone, cyclophosphamide, etoposide, cytarabine,cisplatin, thalidomide, prednisone, thalidomide, bortezomib,lenalidomide, sorafenib, romidepsin or combinations thereof.
 36. Themethod of claim 32, wherein the cytotoxic agent is antibody based.
 37. Amethod for treating a subject having a condition that would benefit fromthe suppression of myeloma cell survival, the method comprising: (a)providing at least one immunoconjugate comprising a targeting antibodyand an effector molecule, wherein the effector is linked to thetargeting antibody via a cleavable linker, and wherein saidimmunoconjugate targets CD138 expressing cells, wherein said targetingantibody comprises: heavy chain variable region CDR3 comprising aminoacid residues 99 to 111 of SEQ ID NO: 1, and light chain variable regionCDR3 comprising amino acid residues 89 to 97 of SEQ ID NO: 2,respectively, and heavy chain variable region CDR1 and CDR2 comprisingamino acid residues 31 to 35 and 51 to 68 of SEQ ID NO: 1, and lightchain variable region CDR1 and CDR 2 comprising amino acid residues 24to 34 and 50 to 56 of SEQ ID NO: 2, respectively; and (b) administeringthe immunoconjugate to the subject to selectively decrease survival orgrowth of said myeloma cells of said subject.
 38. A pharmaceuticalcomposition comprising the immunoconjugate comprising a targetingantibody and an effector molecule, wherein the effector is linked to thetargeting antibody via a cleavable linker, and wherein saidimmunoconjugate targets CD138 expressing cells, wherein said targetingantibody comprises: heavy chain variable region CDR3 comprising aminoacid residues 99 to 111 of SEQ ID NO: 1, and light chain variable regionCDR3 comprising amino acid residues 89 to 97 of SEQ ID NO: 2,respectively, and heavy chain variable region CDR1 and CDR2 comprisingamino acid residues 31 to 35 and 51 to 68 of SEQ ID NO: 1, and lightchain variable region CDR1 and CDR 2 comprising amino acid residues 24to 34 and 50 to 56 of SEQ ID NO: 2, respectively, and one or morepharmaceutically acceptable excipients and further comprising at leastone cytotoxic agent.
 39. (canceled)
 40. The pharmaceutical compositionof claim 38, wherein the cytotoxic agent is mephalan, vincristine,doxorubicin, dexamethasone, cyclophosphamide, etoposide, cytarabine,cisplatin, thalidomide, prednisone, thalidomide, bortezomib,lenalidomide, sorafenib, romidepsin or combinations thereof.
 41. Thepharmaceutical composition of claim 38, wherein the cytotoxic agent isantibody based.
 42. A kit comprising, in separate containers,pharmaceutical compositions for use in combination to inhibit, delayand/or prevent the growth of tumors and/or spread of tumor cells,wherein one container comprises an effective amount of theimmunoconjugate of the pharmaceutical composition of claim 38, andwherein, a separate container comprises a second pharmaceuticalcomposition comprising an effective amount of said cytotoxic agent, forthe inhibition, delay and/or prevention of the growth of tumors and/orspread of tumor cells, and one or more pharmaceutically acceptableexcipients.
 43. The kit of claim 42, wherein said agent in said secondpharmaceutical composition is a selected from the group consisting ofmephalan, vincristine, doxorubicin, dexamethasone, cyclophosphamide,etoposide, cytarabine, cisplatin, thalidomide, prednisone, thalidomide,bortezomib, lenalidomide sorafenib, romidepsin and combinations thereofor is antibody based.
 44. (canceled)
 45. The method of claim 16, whereinsaid effector molecule is sterically hindered and a growth of a tumorinhibiting activity of an unhindered counterpart of the immunoconjugatecomprising a non-cleavable linker exceeds that of the growth of a tumorinhibiting activity of its unhindered counterpart comprising a cleavablelinker, by at least about 5%, at least about 10%, up to about 15%. 46.(canceled)
 47. The method of claim 16, wherein said effector molecule isDM4.
 48. (canceled)
 49. A method of claim 16, wherein said effectormolecule is sterically hindered and wherein the immunoconjugate isadministered to said subject in multiple doses, wherein the soadministered immunoconjugate provides a growth of a tumor inhibitingactivity that exceeds that of its unhindered counterpart by about 20%,about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about90% or more.
 50. (canceled)
 51. (canceled)
 52. The method of claim 49,wherein said multiple doses are administered at intervals of 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 hours, 1, 2,3, 4, 5, 6, 7 days, 1, 2, 3, 4, 5, 6, 7 or 8 weeks.
 53. (canceled)
 54. Amethod for diminishing an amount of cells in direct or indirect contactwith CD138 expressing tumor cells in a subject in need thereofcomprising: administering to said subject at least one immunoconjugatecomprising a targeting antibody and an effector molecule, wherein theeffector is linked to the targeting antibody via a cleavable linker, andwherein said immunoconjugate targets CD138 expressing cells, whereinsaid targeting antibody comprises: heavy chain variable region CDR3comprising amino acid residues 99 to 111 of SEQ ID NO: 1, and lightchain variable region CDR3 comprising amino acid residues 89 to 97 ofSEQ ID NO: 2, respectively, and heavy chain variable region CDR1 andCDR2 comprising amino acid residues 31 to 35 and 51 to 68 of SEQ ID NO:1, and light chain variable region CDR1 and CDR 2 comprising amino acidresidues 24 to 34 and 50 to 56 of SEQ ID NO: 2, respectively in anamount effective to diminish the amount of said cells in direct orindirect contact with said CD138 expressing tumor cells.
 55. The methodof claim 54, wherein said cells in direct or indirect contact with CD138expressing tumor cells consist of cells expressing CD138heterogeneously, non CD138 expressing cells and/or cells beinginaccessible to an effective amount of said at least oneimmunoconjugate.
 56. The method of claim 54, wherein said cells arecells in direct contact with or attached to CD138 expressing tumorcells.
 57. (canceled)